Humanized antibody targeting human CD33 and application thereof

Abstract

The invention discloses a humanized antibody targeting human CD33 and application thereof, and belongs to the field of biological medicine. The invention provides a monoclonal antibody targeting human CD33, and also provides a composition containing the monoclonal antibody, a multispecific antibody, a chimeric antigen receptor and an antibody conjugate. The monoclonal antibody targeting human CD33 has the characteristics of high affinity and strong killing effect on target cells, and is beneficial to prediction, diagnosis and treatment monitoring of related diseases such as myelogenous leukemia.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and relates to humanized antibodies targeting human CD33 and their applications, further relating to humanized antibodies targeting human CD33 and their applications in myeloid leukemia, specifically relating to the construction of CAR-T cell drugs expressing humanized CD33 monoclonal antibodies and the application of antibody-drug conjugates in the killing of Molm13-Luciferase cells. Background Technology

[0002] CD33 is a single-transmembrane protein highly expressed in myeloid cells. It is highly expressed in acute myeloid leukemia progenitor cells but not in normal stem cells. According to existing research, CD33 is mainly distributed in medulloblasts, monocytes, and myeloid cells, with very limited expression in non-hematopoietic tissues, but higher expression levels in myeloid leukemia cells. During the differentiation of pluripotent hematopoietic stem cells, the expression level of CD34 decreases, followed by the expression of CD33. [1] This makes the molecule a highly desirable therapeutic target for myeloid leukemia. Gemtuzumab ozogamicin (disclosed in patent US7727968B2) is a humanized monoclonal IgG4 antibody targeting human CD33. Through conjugation with the cytotoxic calicheamicin derivative, it binds to CD33-expressing leukemia blast cells and is internalized during linker hydrolysis. The released toxin binds to DNA, causing site-specific double-strand breaks, leading to cell death. Chimeric antigen receptor T cells constructed based on this antibody have shown significant killing effects on CD33-expressing myeloid leukemia cells. [2] In addition, combining antibodies targeting human CD33 with agonist antibodies targeting human CD33 to form a bispecific antibody fusion protein, which, after bridging T cells, can also significantly kill myeloid leukemia cells. [3] This invention obtains a monoclonal antibody that specifically binds to human CD33 by immunizing mice, and then further humanizes it; chimeric antigen receptor T cells constructed based on the humanized antibody and conjugated with MMAE toxin both exhibit significant killing effects on tumor cells.

[0003] References

[0004] 1.Andrews, RG, JWSinger, and IDBernstein, Precursors of colony-forming cells in humans can be distinguished from colony-forming cells by expression of the CD33 and CD34 antigens and light scatter properties. The Journal of experimental medicine, 1989.169(5):p.1721-1731.

[0005] 2.Kenderian, SS, et al., CD33-specific chimeric antigen receptor Tcells exhibit potent preclinical activity against human acute myeloidleukemia. Leukemia, 2015.29(8):p.1637-47.

[0006] 3. Hoseini, SS, et al., T cell engaging bispecific antibodies targeting CD33 IgV and IgC domains for the treatment of acute myeloid leukemia. JImmunother Cancer, 2021.9(5). Summary of the Invention

[0007] The problem the invention aims to solve

[0008] Based on the problems existing in the prior art, this invention screened out monoclonal antibodies that can specifically bind to CD33, further constructed CAR-T cells expressing humanized CD33 monoclonal antibodies, evaluated their killing effect on Molm13-Luciferase cells, and also prepared antibody conjugates and evaluated their effects on target cells in vivo and in vitro.

[0009] Solution for solving the problem

[0010] A first aspect of the invention provides an antibody or antigen-binding fragment thereof targeting CD33, comprising a heavy chain variable region and a light chain variable region, wherein,

[0011] The light chain variable region includes:

[0012] LCDR1 as shown in SEQ ID NO:6, SEQ ID NO:13, SEQ ID NO:20 or SEQ ID NO:27;

[0013] LCDR2, as shown in YAS or YTS;

[0014] LCDR3 as shown in SEQ ID NO:7, SEQ ID NO:14, SEQ ID NO:21, SEQ ID NO:28 or SEQ ID NO:32;

[0015] The heavy chain variable region includes:

[0016] HCDR1 as shown in SEQ ID NO:2, SEQ ID NO:9, SEQ ID NO:16 or SEQ ID NO:23;

[0017] HCDR2 as shown in SEQ ID NO:3, SEQ ID NO:10, SEQ ID NO:17, SEQ ID NO:24 or SEQ ID NO:30;

[0018] HCDR3, as shown in SEQ ID NO:4, SEQ ID NO:11, SEQ ID NO:18, or SEQ ID NO:25.

[0019] In some embodiments, the light chain variable region includes LCDR1, LCDR2, and LCDR3 selected from any one of (a1) to (a5):

[0020] (a1) LCDR1 as shown in SEQ ID NO:6, LCDR2 as shown in YAS, or LCDR3 as shown in SEQ ID NO:7;

[0021] (a2) LCDR1 as shown in SEQ ID NO:13, LCDR2 as shown in YTS, or LCDR3 as shown in SEQ ID NO:14;

[0022] (a3) LCDR1 as shown in SEQ ID NO:20, LCDR2 as shown in YTS, or LCDR3 as shown in SEQ ID NO:21;

[0023] (a4) LCDR1 as shown in SEQ ID NO:27, LCDR2 as shown in YTS, or LCDR3 as shown in SEQ ID NO:28;

[0024] (a5) LCDR1 as shown in SEQ ID NO:13, LCDR2 as shown in YTS, or LCDR3 as shown in SEQ ID NO:32;

[0025] The heavy chain variable region includes HCDR1, HCDR2 and HCDR3 selected from any of the following (b1) to (b5):

[0026] (b1) HCDR1 as shown in SEQ ID NO:2, HCDR2 as shown in SEQ ID NO:3, or HCDR3 as shown in SEQ ID NO:4;

[0027] (b2) HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, or HCDR3 as shown in SEQ ID NO:11;

[0028] (b3) HCDR1 as shown in SEQ ID NO:16, HCDR2 as shown in SEQ ID NO:17, or HCDR3 as shown in SEQ ID NO:18;

[0029] (b4) HCDR1 as shown in SEQ ID NO:23, HCDR2 as shown in SEQ ID NO:24, or HCDR3 as shown in SEQ ID NO:25;

[0030] (b5) HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:30, or HCDR3 as shown in SEQ ID NO:11.

[0031] In some embodiments, the antibody targeting CD33 or its antigen-binding fragment includes a murine antibody or a fragment thereof, a chimeric antibody or a fragment thereof, a humanized antibody or a fragment thereof, and / or a fully human antibody or a fragment thereof.

[0032] In some embodiments, the light chain variable region of the murine antibody or fragment thereof comprises: a sequence shown in any one of SEQ ID NO: 5, 12, 19, 26 and 31, or a sequence having at least 97%, 98% or 99% identity with any one of SEQ ID NO: 5, 12, 19, 26 and 31;

[0033] The heavy chain variable region of the murine antibody or its fragment comprises: a sequence shown in any one of SEQ ID NO: 1, 8, 15, 22 and 29, or a sequence having at least 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with any one of SEQ ID NO: 1, 8, 15, 22 and 29.

[0034] In some embodiments, the light chain variable region of the humanized antibody or fragment thereof comprises: a sequence as shown in any one of SEQ ID NO:34, 36, 38, 39 and 42, or a sequence having at least 97%, 98% or 99% identity with any one of SEQ ID NO:34, 36, 38, 39 or 42;

[0035] The heavy chain variable region of the humanized antibody or its fragment comprises: a sequence as shown in any one of SEQ ID NO:33, 35, 37, 40 and 41, or a sequence having at least 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with any one of SEQ ID NO:33, 35, 37, 40 and 41.

[0036] In some embodiments, the antibody targeting CD33 or its antigen-binding fragment comprises a heavy chain constant region of human IgG1 or a variant thereof, human IgG2 or a variant thereof, human IgG3 or a variant thereof, or human IgG4 or a variant thereof.

[0037] And / or,

[0038] It contains light chain constant regions derived from human κ chain, λ chain or variants thereof.

[0039] In some embodiments, the antigen-binding fragment is selected from at least one of F(ab)2, Fab', Fab, Fv, scFv, multispecific antibodies, and single-chain antibodies.

[0040] A second aspect of the present invention provides a chimeric antigen receptor comprising:

[0041] (A) An extracellular domain that specifically binds to CD33;

[0042] (B) Transmembrane domain;

[0043] (C) Intracellular signal transduction domains;

[0044] Preferably, the extracellular domain comprises an antibody or antigen-binding fragment thereof targeting CD33 as described above;

[0045] More preferably, the chimeric antigen receptor comprises a sequence as shown in any one of SEQ ID NO:45-49, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO:45-49.

[0046] A third aspect of the invention provides an isolated polynucleotide, wherein the polynucleotide encodes an antibody targeting CD33 as described above, or an antigen-binding fragment thereof, or a chimeric antigen receptor as described above.

[0047] A fourth aspect of the present invention provides an expression cassette, wherein the expression cassette comprises the polynucleotides described above.

[0048] A fifth aspect of the present invention provides an expression vector, wherein the expression vector contains the polynucleotides as described above or the expression cassettes as described above.

[0049] A sixth aspect of the present invention provides a host cell, wherein the host cell comprises the polynucleotide, expression cassette, or expression vector as described above.

[0050] A seventh aspect of the present invention provides a recombinant immune cell, wherein the recombinant immune cell contains a chimeric antigen receptor as described above.

[0051] In some embodiments, the immune cells are immune cells derived from mammals;

[0052] Optionally, the immune cells are selected from one or more of T cells, B cells, NK cells, mast macrophages, and tumor-infiltrating lymphocytes;

[0053] Preferably, the immune cells are selected from T cells, NK cells, or macrophages;

[0054] More preferably, the T cells are selected from CD4. + CD8 + T cells, CD8 + T cells, CD4 + T cells, effector T cells, suppressor T cells, primitive T cells, memory T cells, γ-δ T cells, α-β T cells, CD4+ - CD8 - One or more of double-negative T cells and NKT cells.

[0055] The present invention provides the use of the antibody or antigen-binding fragment thereof targeting CD33 as described above, the chimeric antigen receptor as described above, the polynucleotide as described above, the expression cassette as described above, the expression vector as described above, the host cell as described above, or the recombinant immune cell as described above in the preparation of a medicament for treating tumors.

[0056] Optionally, the tumor is CD33 positive;

[0057] Preferably, the tumor is cancer.

[0058] The present invention also provides a multispecific antibody, wherein the multispecific antibody comprises an antibody or antigen-binding fragment thereof targeting CD33 as described above.

[0059] The present invention provides a composition comprising at least one of the following: an antibody targeting CD33 as described above or an antigen-binding fragment thereof, a chimeric antigen receptor as described above, a polynucleotide as described above, an expression cassette as described above, an expression vector as described above, a host cell as described above, and a recombinant immune cell as described above.

[0060] Optionally, the composition comprises a pharmaceutical composition;

[0061] Optionally, the pharmaceutical composition contains a pharmaceutically acceptable carrier and / or excipients.

[0062] The present invention provides a detection or diagnostic reagent or kit comprising: an antibody or antigen-binding fragment thereof targeting CD33 as described above.

[0063] This invention provides a method for detecting CD33 protein in a sample in vitro without diagnostic purposes, comprising the following steps:

[0064] (a) In vitro, the sample is contacted with an antibody or antigen-binding fragment thereof that targets CD33 as described above;

[0065] (b) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of CD33 protein in the sample.

[0066] The present invention provides an antibody conjugate comprising an antibody or antigen-binding fragment thereof targeting CD33 as described above;

[0067] Optionally, the antibody conjugate contains a cytotoxic drug;

[0068] Optionally, the cytotoxic drug is monomethylauratestatin (MMAE).

[0069] The effects of the invention

[0070] The antibody targeting CD33 provided by this invention has high affinity and can bind to mammalian cells expressing CD33.

[0071] The antibodies, chimeric antigen receptors, and antibody conjugates targeting CD33 provided by this invention have a good killing effect on target cells and inhibit tumor growth.

[0072] The method for detecting CD33 protein in in vitro non-diagnostic samples, and the detection or diagnostic kit containing antibodies targeting CD33 provided by the present invention can also effectively detect CD33 protein. Attached Figure Description

[0073] Figure 1 The image shows the FACS detection results of the candidate clone binding to the target protein.

[0074] Figure 2 The results of affinity testing of the prepared humanized antibody are shown.

[0075] Figure 3 The image shows the results of the detection of target cell killing by chimeric antigen receptor T cells constructed from candidate-derived antibodies.

[0076] Figure 4 The results of CAR-T cytokine secretion assay are shown.

[0077] Figure 5 The results of CAR-T in vivo efficacy testing are shown.

[0078] Figure 6 The results show the detection results of the killing effect of antibody-conjugated toxin on target cells.

[0079] Figure 7 The results show the killing effect of antibody-conjugated toxin on tumor cells in mice.

[0080] Figure 8 The image shows the effect of antibody-drug conjugate on the survival time of mice.

[0081] Figure 9 The image shows the effect of antibody-drug conjugate on mouse body weight. Detailed Implementation

[0082] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0083] In this invention, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0084] In this invention, "optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes both cases in which the event occurs and cases in which the event does not occur.

[0085] In this invention, the terms "some specific / preferred embodiments," "other specific / preferred embodiments," "embodiment," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one embodiment described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0086] In this specification, the terms "preferred" and "ideal" are not intended to limit the scope of the claimed invention, or to imply that certain features are critical, necessary, or even important to the structure or function of the claimed invention. Rather, these terms are merely used to emphasize alternative or additional features that may or may not be used in specific embodiments of the invention.

[0087] In this invention, unless otherwise specified, the term "comprising" as used herein can be open-ended or closed-ended. For example, "comprising" may mean that it may also include other components not listed, or it may only include the listed components.

[0088] In this invention, the term "CD33" refers to CD33, a member of the sialic acid adhesin family, which is expressed on cells of the hematopoietic lineage (including myeloid precursors, monocytes, macrophages, dendritic cells, and mast cells) and on tumor cells and leukemia stem cells associated with myeloproliferative or mast cell proliferative disorders (including acute myeloid leukemia and myelodysplastic syndrome).

[0089] The "antibody" described in this invention is an immunoglobulin. A complete antibody is typically a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain differ, thus their antigenicity also differs. Based on this, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, differences in the amino acid composition of its hinge region and the number and position of disulfide bonds in the heavy chain can further divide it into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chains are classified as κ or λ chains based on differences in their constant regions. Each of the five classes of Ig can have either a κ or λ chain.

[0090] The sequence of approximately 110 amino acids near the N-terminus of both the antibody heavy and light chains varies considerably and is known as the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are called the constant region. The variable region includes three hypervariable regions (HVR) and four relatively conserved backbone regions (FR). The three hypervariable regions determine the antibody's specificity and are also called complementarity-determining regions (CDR). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

[0091] In this specification, the term "antibody framework" or "FR region" refers to a portion of the variable domain VL or VH that serves as a scaffold for the antigen-binding loop (CDR) of that variable domain. Essentially, it is a variable domain without a CDR.

[0092] In this specification, the terms "complementarity-determining region," "CDR," or "hypervariant region" refer to one of the six hypervariable regions within the variable domain of an antibody that primarily facilitate antigen binding. Typically, each heavy chain variable region contains three CDRs (HCDR1, HCDR2, HCDR3), and each light chain variable region contains three CDRs (LCDR1, LCDR2, LCDR3). The amino acid sequence boundaries of CDRs can be determined using any of a variety of well-known schemes, including the “Kabat” numbering rule (see Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the “Chothia” numbering rule (see Al-Lazikani et al., (1997) JMB 273: 927-948), and the ImMunoGenTics (IMGT) numbering rule (Lefranc M.P., Immunologist, 7, 132-136 (1999); Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003), etc.).

[0093] The antibodies of this invention include murine antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies. In addition to full-length antibodies, the antibodies of this invention also include antigen-binding fragments capable of binding to antigens.

[0094] In this invention, the term "mouse antibody" refers to a monoclonal antibody derived from a mouse and prepared in accordance with the knowledge and skills in the art. Preparation involves injecting an antigen into the test subject, followed by isolating a hybridoma expressing an antibody with the desired sequence or functional characteristics. When the injected test subject is a mouse, the resulting antibody is a mouse antibody.

[0095] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody. It can mitigate the immune response induced by murine antibodies. To create a chimeric antibody, a hybridoma secreting a murine-specific monoclonal antibody is first established. Then, the variable region gene is cloned from the murine hybridoma cells. Next, the constant region gene of the human antibody is cloned as needed. The murine variable region gene and the human constant region gene are ligated to form a chimeric gene, which is then inserted into an expression vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic system. The light chain of the chimeric antibody further includes the constant region of a human κ, λ chain, or a variant thereof. The heavy chain of the chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or variants thereof, preferably comprising a heavy chain constant region of human IgG1, IgG2 or IgG4, or using a variant of the heavy chain constant region of IgG1, IgG2 or IgG4 with an amino acid mutation (such as YTE mutation or reversion mutation, L234A and / or L235A mutation, or S228P mutation).

[0096] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody produced by grafting a mouse CDR sequence into the variable region framework of a human antibody, i.e., a human germline antibody framework sequence of different types. This overcomes the heterologous response induced by chimeric antibodies carrying a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases or publicly available references that include germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available at www.mrccpe.com.ac.uk / vbase) and in Kabat, E.A. et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition. To avoid a decrease in activity along with a decrease in immunogenicity, the human antibody variable region framework sequence can be subjected to minimal reverse or reversion mutations to maintain activity. The humanized antibodies of this invention also include humanized antibodies further characterized by affinity maturation mutations of the CDR by yeast.

[0097] The term "fully human antibody" or "human antibody" refers to an antibody having variable regions in which both the FR and CDR are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, that constant region is also derived from a human germline immunoglobulin sequence. Fully human antibodies described herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced through random or site-specific mutagenesis in vitro or through somatic mutations in vivo). However, the term "fully human antibody" herein is not intended to include antibodies in which a CDR sequence derived from another mammalian species (e.g., mouse) has been grafted onto a human frame sequence.

[0098] Due to contact residues with the antigen, CDR transplantation can lead to a decrease in the affinity of the resulting antibody or its antigen-binding fragment for the antigen due to the framework residues in contact with the antigen. Such interactions may be a result of high somatic mutations. Therefore, it may still be necessary to transplant such donor framework amino acids into the framework of humanized antibodies. The amino acid residues involved in antigen binding from non-human antibodies or their antigen-binding fragments can be identified by examining the variable region sequence and structure of animal monoclonal antibodies. Residues in the CDR donor framework that differ from the species can be considered relevant. If the closest species cannot be determined, the sequence can be compared with a subclass common sequence or a common sequence of animal antibody sequences with a high percentage of similarity. Rare framework residues are thought to be a result of high somatic mutations, thus playing an important role in binding.

[0099] The terms "human antibody" (HuMAb), "human-derived antibody," "fully human antibody," and "completely human antibody" are used interchangeably. A human antibody can be an antibody derived from a genetically modified organism (GMO) that is "engineered" to produce specific human antibodies in response to antigenic stimulation and can be produced by any method known in the art. In some techniques, elements of human heavy and light chain loci are introduced into cell lines derived from embryonic stem cell lines, where endogenous heavy and light chain loci in these cell lines are targeted for disruption. The GMO can synthesize human antibodies specific to human antigens, and the organism can be used to produce human antibody-secreting hybridomas. A human antibody can also be an antibody in which the heavy and light chains are encoded by nucleotide sequences derived from the DNA of one or more individuals. Completely human antibodies can also be constructed using gene or chromosome transfection methods and phage display technology, or from in vitro activated B cells, all of which are known in the art.

[0100] The term "antigen-binding fragment" or "functional fragment" of an antibody refers to one or more fragments that enable the antibody to maintain its specific ability to bind to an antigen. It has been shown that fragments of full-length antibodies can be used for antigen-binding function. Examples of binding fragments included in the term "antigen-binding fragment" of an antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments connected by disulfide bridges on their hinge regions; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VH and VL domains on a single arm of the antibody; (v) dsFv, stable antigen-binding fragments formed by interchain disulfide bonds between VH and VL; and (vi) bispecific, bispecific, and multispecific antibodies containing fragments such as scFv, dsFv, and Fab. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked by synthetic linkers using recombinant methods, thereby enabling the production of a single protein chain (called a single-chain Fv (scFv) in which the VL and VH regions pair to form a monovalent molecule; see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also included in the term "antigen-binding fragment" of an antibody. Such antigen-binding fragments are obtained using conventional techniques known to those skilled in the art, and fragments are screened for functionality in the same manner as for intact antibodies. Antigen-binding moieties can be generated by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. Antibodies can be different types of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0101] Fab is an antigen-binding fragment with a molecular weight of approximately 50,000 and antigen-binding activity obtained by treating IgG antibody molecules with the protease papain (which cleaves amino acid residues of the H chain). Approximately half of the N-terminal side of the H chain and the entire L chain are linked together by disulfide bonds.

[0102] F(ab')2 is an antigen-binding fragment with a molecular weight of approximately 100,000, containing two Fab regions connected at the hinge position, obtained by digesting the portion below the two disulfide bonds in the hinge region of IgG with the enzyme pepsin.

[0103] Fab' is an antigen-binding fragment with a molecular weight of approximately 50,000 and possessing antigen-binding activity, obtained by cleaving the disulfide bonds in the hinge region of the aforementioned F(ab')2. The Fab' of the present invention can be produced by treating F(ab')2 that specifically recognizes and binds to antigens with a reducing agent such as dithiothreitol.

[0104] In addition, the Fab' can be produced by inserting DNA encoding the Fab' fragment of an antibody into a prokaryotic or eukaryotic expression vector and then introducing the vector into a prokaryote or eukaryote to express the Fab'.

[0105] The terms “single-chain antibody,” “single-chain Fv,” or “scFv” refer to molecules containing a variable domain (or region; VH) of the antibody heavy chain and a variable domain (or region; VL) of the antibody light chain linked by a linker. Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeating GGGGS amino acid sequences or variants thereof, for example, using variants with 1–4 repeats (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444–6448). Other connectors that can be used in this invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.

[0106] In this invention, the terms "specific binding" and "selective binding" refer to the binding of an antibody to an epitope on a predetermined antigen.

[0107] In this invention, the term "epitope" refers to a site on an antigen that specifically binds to an immunoglobulin or antibody. Epitopes can be formed from adjacent amino acids or from non-adjacent amino acids arranged side-by-side through the ternary folding of a protein. Epitopes formed from adjacent amino acids are generally retained upon exposure to denaturing solvents, while epitopes formed through ternary folding are generally lost upon treatment with denaturing solvents. Epitopes typically comprise at least 3-15 amino acids in a distinctive spatial conformation. Methods for determining which epitopes bind to a given antibody are well known in the art, including immunoblotting and immunoprecipitation assays. Methods for determining the spatial conformation of epitopes include techniques in the art, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0108] In this invention, the terms “polypeptide,” “protein,” and “peptide” are used interchangeably herein to refer to a polymeric form of amino acids of any length, including encoded and non-coding amino acids, chemically or biochemically modified or derived amino acids, and polypeptides having a similar peptide backbone.

[0109] In this invention, the terms "polynucleotide" and "nucleic acid" are used interchangeably to refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or similar. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. Nucleic acid molecules can be linear or circular.

[0110] In this invention, the three-letter and single-letter codes for amino acids are as described in J.biol.chem, 243, p3558 (1968).

[0111] In this invention, "identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. When positions in two compared sequences are occupied by the same base or amino acid monomer subunit—for example, if every position in two DNA molecules is occupied by adenine—then the molecules are homologous at that position. The percentage of identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, at optimal sequence alignment, if 6 out of 10 positions in two sequences match or are homologous, then the two sequences are 60% homologous. Generally, comparisons are made when the highest percentage of identity is obtained by aligning the two sequences.

[0112] In this specification, the term "codon optimization" refers to the configuration of the nucleotide sequence encoding a polypeptide to contain codons preferred by the host cell or organism in order to improve gene expression and translation efficiency in the host cell or organism.

[0113] In this specification, "expression vector" refers to a vector comprising a recombinant polynucleotide, said recombinant polynucleotide including an expression control sequence operatively linked to a nucleotide sequence to be expressed. The expression vector includes cis-acting components sufficient for expression; other components for expression may be provided by a host cell or in an in vitro expression system. The expression vector includes all those known in the art, such as granules, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that introduce the recombinant polynucleotide. In this invention, the term "CAR-T cell" refers to a chimeric antigen receptor T cell, typically composed of an extracellular targeting domain, a transmembrane region, and an intracellular signaling domain. The CAR targeting domain is typically derived from the antigen-binding region of an antibody, i.e., the immunoglobulin heavy and light chains that form the antibody binding site are responsible for recognizing and binding the target antigen; the transmembrane region is a hinge or spacer region that anchors a single-stranded variable domain to the cell membrane; the intracellular signaling domain consists of a co-stimulatory factor and a CD3 signaling domain. Once the antigen is recognized and bound, a stimulating signal is generated and transmitted to the intracellular signaling domain, activating the T cell and enabling it to perform effector functions.

[0114] In this invention, the term "chimeric antigen receptor (CAR)" refers to a fusion protein comprising an extracellular domain capable of binding an antigen, a transmembrane domain derived from a different polypeptide, and at least one intracellular domain. This enables the anchoring of specific molecules (such as antibodies) that recognize tumor cell surface antigens to immune cells (such as T cells), allowing the immune cells to recognize tumor antigens or viral antigens and kill tumor cells or viral cells. The "extracellular domain capable of binding an antigen" refers to any oligopeptide or polypeptide capable of binding a particular antigen. The "intracellular domain" refers to any oligopeptide or polypeptide known to function as a domain that transmits signals to activate or inhibit intracellular biological processes.

[0115] In this specification, the term "293F cell" refers to a cell line derived from 293 cells through genetic technology. Transient transfection of 293F cells is a convenient way to overexpress proteins and obtain intracellular and extracellular (secreted or membrane) proteins.

[0116] In this specification, “application,” “giving,” and “treatment,” when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. “Application,” “giving,” and “treatment” can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cell treatment includes contact between a reagent and cells, and contact between a reagent and a fluid, wherein the fluid is in contact with the cells. “Application,” “giving,” and “treatment” also mean, by means of a reagent, diagnostic agent, conjugated composition, or by means of another cell in vitro and ex vivo, such as the treatment of cells. “Treatment,” when applied to humans, veterinary, or research subjects, refers to therapeutic, preventative, or prophylactic measures, research, and diagnostic applications.

[0117] In this invention, the term "treatment" refers to a method of alleviating or eliminating symptoms and / or accompanying symptoms.

[0118] In this invention, the term "tumor" refers to a growth or solid lesion formed by abnormal cell growth, and optionally, the tumor cells are Molm13-Luciferase.

[0119] In this invention, the term "transfection" refers to the entry of recombinant plasmid vectors or free nucleotides into eukaryotic cells mediated by liposomes or the like.

[0120] In this invention, the term "infection" refers to the process by which a recombinant viral vector invades recipient cells in gene transfer experiments. CAR-T cells bind to specific antigens via CARs, thereby transmitting signals into the cell and activating the cell. The activation of CAR-expressing cells varies depending on the type of host cell and the intracellular domains of the CAR, and can be confirmed based on indicators such as the release of cytokines, improved cell proliferation, and changes in cell surface molecules. For example, the cytotoxicity of cytokines released from activated cells (tumor necrosis factor, lymphotoxin, etc.) can cause destruction of cells expressing the target antigen. Furthermore, the release of cytokines or changes in cell surface molecules can stimulate other immune cells, such as B cells, dendritic cells, NK cells, and macrophages.

[0121] In this specification, the term "tag" refers to a short peptide that is fused or linked to a target protein (e.g., the antibody of the present invention) and thereby facilitates the soluble expression, detection, and / or purification of the recombinant protein. The tag may be fused to or linked to the N-terminus and / or C-terminus of the target protein (optionally via a linker or protease cleavage site).

[0122] In this specification, the terms “cell,” “cell line,” and “cell culture” are used interchangeably, and all such names include progeny. Therefore, the terms “transformant” and “transformed cell” include primary test cells and cultures derived therefrom, regardless of the number of transfections. It should also be understood that, due to intentional or unintentional mutations, all progeny may not be exactly identical in DNA content. This includes mutant progeny with the same function or biological activity as those screened from the original transformed cells.

[0123] In this invention, "multispecific antibody" includes the antibody targeting CD33 described above, or its antigen-binding fragment. The multispecific antibody also includes antigen-binding fragments targeting other antigens.

[0124] In this specification, "diagnosis" includes the detection or identification of a subject's disease state or condition, determining the likelihood that a subject will have a given disease or condition, determining the likelihood that a subject with a disease or condition will respond to treatment, determining the prognosis (or possible progression or regression) of a subject with a disease or condition, and determining the effect of treatment on a subject with a disease or condition.

[0125] In this specification, "treatment" means administering an oral or topical therapeutic agent, such as an antibody comprising any of the present invention, to a patient who has one or more symptoms of a disease, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, a therapeutic agent is administered in a treated patient or population in an amount that effectively relieves one or more symptoms of a disease, whether by inducing the regression of such symptoms or inhibiting their progression to any clinically measurable degree. The amount of a therapeutic agent that effectively relieves any specific disease symptom (also referred to as a "therapeuticly effective amount") can vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been relieved can be evaluated using any clinical testing method commonly used by a physician or other healthcare professional to assess the severity or progression of the symptoms.

[0126] In this specification, "pharmaceutical composition" means containing one or more antibodies described herein, as well as other components such as physiological / pharmaceutical-grade carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertment of its biological activity.

[0127] In this specification, the term "pharmaceutical acceptable" (or "pharmacologically acceptable") means a molecular entity and composition that, when appropriate, does not produce an adverse reaction, allergic reaction, or other adverse reaction when administered to animals or humans. As used herein, the term "pharmaceutical acceptable carrier" includes any and all solvents, dispersion media, coatings, antimicrobial agents, isotonic agents and absorption delay agents, buffers, excipients, binders, lubricants, gels, surfactants, etc., that can be used as a medium for pharmaceutically acceptable substances.

[0128] The technical solution of the present invention will be described in detail below:

[0129] <Antibodies or antigen-binding fragments targeting CD33>

[0130] According to some embodiments of the present invention, an antibody or antigen-binding fragment thereof targeting CD33 is provided, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region or a light chain variable region, both the heavy chain variable region and the light chain variable region comprising three complementarity-determining regions (CDRs): LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3.

[0131] In some embodiments, the LCDR1 comprises an amino acid sequence as shown in SEQ ID NO:6 or SEQ ID NO:13 or SEQ ID NO:20 or SEQ ID NO:27, or a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% or higher sequence identity with any one of SEQ ID NO:6 or SEQ ID NO:13 or SEQ ID NO:20 or SEQ ID NO:27.

[0132] In some embodiments, the LCDR2 comprises an amino acid sequence such as YAS or YTS, or a variant thereof having at least 70% or 99% or higher sequence identity with either YAS or YTS.

[0133] In some embodiments, the LCDR3 comprises amino acid sequences as shown in SEQ ID NO:7, SEQ ID NO:14, SEQ ID NO:21, SEQ ID NO:28, SEQ ID NO:32, or variants thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% or higher sequence identity with any one of SEQ ID NO:7, SEQ ID NO:14, SEQ ID NO:21, SEQ ID NO:28, SEQ ID NO:32.

[0134] In some embodiments, the HCDR1 comprises an amino acid sequence as shown in SEQ ID NO:2, SEQ ID NO:9, SEQ ID NO:16 or SEQ ID NO:23, or a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% or higher sequence identity with any one of SEQ ID NO:2, SEQ ID NO:9, SEQ ID NO:16 or SEQ ID NO:23.

[0135] In some embodiments, the LCDR2 comprises an amino acid sequence as shown in SEQ ID NO:3, SEQ ID NO:10, SEQ ID NO:17, SEQ ID NO:24 or SEQ ID NO:30, or a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% or higher sequence identity with any one of SEQ ID NO:3, SEQ ID NO:10, SEQ ID NO:17, SEQ ID NO:24 and SEQ ID NO:30.

[0136] In some embodiments, the LCDR3 comprises an amino acid sequence as shown in SEQ ID NO:4, SEQ ID NO:11, SEQ ID NO:18, or SEQ ID NO:25, or a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% or higher sequence identity with any one of SEQ ID NO:4, SEQ ID NO:11, SEQ ID NO:18, and SEQ ID NO:25.

[0137] Furthermore, the antibody targeting CD33 or its antigen-binding fragment, wherein the light chain variable region comprises:

[0138] (a1) LCDR1 as shown in SEQ ID NO:6, LCDR2 as shown in SEQ ID NO:7, and LCDR3 as shown in SEQ ID NO:8;

[0139] (a2) LCDR1 as shown in SEQ ID NO:13, LCDR2 as shown in sequence YTS, and CDR3 as shown in SEQ ID NO:14;

[0140] (a3) LCDR1 as shown in SEQ ID NO:20, LCDR2 as shown in sequence YTS, and LCDR3 as shown in SEQ ID NO:21;

[0141] (a4) LCDR1 as shown in SEQ ID NO:27, LCDR2 as shown in sequence YTS, and LCDR3 as shown in SEQ ID NO:28;

[0142] (a5) LCDR1 as shown in SEQ ID NO:13, LCDR2 as shown in sequence YTS, and LCDR3 as shown in SEQ ID NO:32;

[0143] Its heavy chain variable region includes:

[0144] (b1) HCDR1 as shown in SEQ ID NO:2, HCDR2 as shown in SEQ ID NO:3, and HCDR3 as shown in SEQ ID NO:4;

[0145] (b2) HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and CDR3 as shown in SEQ ID NO:11;

[0146] (b3) HCDR1 as shown in SEQ ID NO:16, HCDR2 as shown in SEQ ID NO:17, and HCDR3 as shown in SEQ ID NO:18;

[0147] (b4) HCDR1 as shown in SEQ ID NO:23, HCDR2 as shown in SEQ ID NO:24, and HCDR3 as shown in SEQ ID NO:25;

[0148] (b5) HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:30, and HCDR3 as shown in SEQ ID NO:11.

[0149] In some alternative embodiments, the antibody targeting CD33 or its antigen-binding fragment includes a murine antibody or a fragment thereof, a chimeric antibody or a fragment thereof, a humanized antibody or a fragment thereof, and / or a fully human antibody or a fragment thereof.

[0150] In some exemplary embodiments, LCDR1, LCDR2, LCDR3 or HCDR1, HCDR2 and HCDR3 are separated by frame regions FR1, FR2, FR3 and FR4.

[0151] In some alternative implementations, the frame region FR is of human, mouse, rabbit, or camel origin.

[0152] In some preferred embodiments, the frame region FR is a mouse-derived FR region.

[0153] In some alternative embodiments, the antibody targeting CD33 or its antigen-binding fragment comprises a murine antibody or a fragment thereof, wherein the light chain variable region of the murine antibody or its fragment comprises one or more of the following (i) to (iii):

[0154] (i) The amino acid sequences shown in SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:19, SEQ ID NO:26, and SEQ ID NO:31;

[0155] (ii) A sequence having at least 97%, 98%, or 99% identity among any of the amino acid sequences shown in SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:19, SEQ ID NO:26, or SEQ ID NO:31;

[0156] (iii) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted in the amino acid sequences shown in SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:19, SEQ ID NO:26, and SEQ ID NO:31, and retains the function of the amino acid sequences shown in SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:19, SEQ ID NO:26, and SEQ ID NO:31.

[0157] The heavy chain variable region of the murine antibody or its fragment contains one or more of the following (iv) to (vi):

[0158] (iv) any of the sequences shown in SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:15, SEQ ID NO:22, or SEQ ID NO:29;

[0159] (v) A sequence having at least 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with any one of SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:15, SEQ ID NO:22 or SEQ ID NO:29;

[0160] (vi) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted in the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:15, SEQ ID NO:22, and SEQ ID NO:29, and retains the function of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:15, SEQ ID NO:22, and SEQ ID NO:29.

[0161] In some other preferred embodiments, the frame region FR includes a human FR region. Optionally, the antibody targeting CD33 or its antigen-binding fragment is partially or fully humanized.

[0162] In some alternative embodiments, the antibody targeting CD33 or its antigen-binding fragment comprises a humanized antibody or a fragment thereof, wherein the light chain variable region of the humanized antibody or its fragment comprises one or more of the following (vii) to (ix):

[0163] (vii) Amino acid sequences as shown in SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:42;

[0164] (viii) A sequence having at least 97%, 98%, or 99% identity among any of the amino acid sequences shown in SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:42;

[0165] (ix) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted in the amino acid sequences shown in SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:42, and retains the function of the amino acid sequences shown in SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:42.

[0166] The heavy chain variable region of the humanized antibody or its fragment contains one or more of the following (x) to (xii):

[0167] (x) The sequence shown in any one of SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:40, or SEQ ID NO:41;

[0168] (xi)A sequence having at least 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with any one of SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:41;

[0169] (xii) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted in the amino acid sequences shown in SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:40, and SEQ ID NO:41, and retains the function of the amino acid sequences shown in SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:40, and SEQ ID NO:41.

[0170] In some embodiments, the antibody targeting CD33 or its antigen-binding fragment further comprises a heavy chain constant region derived from human IgG1, human IgG2, human IgG3, human IgG4 or variants thereof; and / or, the antibody targeting CD33 or its antigen-binding fragment further comprises a light chain constant region derived from human κ chain, λ chain or variants thereof.

[0171] In some embodiments, the antigen-binding fragment is selected from at least one of F(ab)2, Fab', Fab, Fv, scFv, multispecific antibodies, and single-chain antibodies.

[0172] The antibody targeting CD33 or its antigen-binding fragment of the present invention refers to a polypeptide having CD33 binding activity and including the aforementioned CDR region. This term also includes variants of polypeptides containing the aforementioned CDR region that have the same function as the antibody targeting CD33 or its antigen-binding fragment of the present invention. These variants include, but are not limited to: deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically less than 20, preferably less than 10, more preferably less than 5) to the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, adding one or more amino acids to the C-terminus and / or N-terminus generally does not alter the function of the protein. Variations of single-domain antibodies include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibodies of the present invention under high or low severity conditions, and polypeptides or proteins obtained using antiserum of the antibody targeting CD33 of the present invention or its antigen-binding fragment.

[0173] Chimeric antigen receptors

[0174] This invention provides a chimeric antigen receptor comprising:

[0175] (A) An extracellular domain that specifically binds to CD33;

[0176] (B) Transmembrane domain;

[0177] (C) Intracellular signal transduction domains.

[0178] (Extracellular domain)

[0179] In this invention, the extracellular domain is the extracellular antigen-binding domain, which consists of an antigen-binding domain (ABD, scFv) and a hinge region that serves as a connector.

[0180] In some implementations, the scFv is a single-chain variable fragment of a monoclonal antibody that recognizes and binds to an antigen.

[0181] In some embodiments, the antigen-binding domain comprises the CD33-binding antibody or its antigen-binding fragment as described above. Exemplarily, the antigen-binding domain comprises the humanized antibody or its fragment as described above.

[0182] In this invention, the "hinge region" is a structural domain that makes up IgG, IgA, and IgD immunoglobulin molecules. Located between CH1 and CH2, it connects the Fab and Fc segments, or connects the extracellular functional domains of membrane proteins such as CD8, CD4, and PD1 to their transmembrane regions. This segment is rich in proline, does not form α-helices, and is prone to stretching and some degree of twisting, which facilitates complementary binding between the antibody's antigen-binding site and the antigen epitope.

[0183] (Transmembrane domain)

[0184] In this invention, the term "transmembrane domain" (also referred to as a transmembrane region) may include one or more amino acid fragments adjacent to the transmembrane region, such as one or more amino acids associated with an extracellular region of the protein from which the transmembrane protein originates (e.g., amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids from the extracellular region). In one aspect, the transmembrane domain is a domain associated with one of the other domains of the chimeric receptor; for example, in one embodiment, the transmembrane domain may originate from the same protein from which the signal transduction domain, costimulatory domain, or hinge domain originates. In some cases, the transmembrane domain may be selectively or by amino acid substitution to prevent such a domain from binding to transmembrane domains of the same or different surface membrane proteins, for example, to minimize interactions with other members of the receptor complex containing the transmembrane domain. In one aspect, the transmembrane domain is capable of isodimerizing with another chimeric receptor on the cell surface expressing the chimeric receptor. In one aspect, the amino acid sequence of the transmembrane domain may be modified or substituted to minimize interaction with the binding domain of the natural binding partner present in cells expressing the same chimeric receptor. The transmembrane domain may be derived from a natural or recombinant source. When the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. In one aspect, the transmembrane domain is capable of signaling to the intracellular domain whenever the chimeric receptor binds to the target antigen. The transmembrane domains particularly used in this invention may include at least the following transmembrane domains: for example, the α, β, or ζ chain of the T-cell receptor, and the transmembrane domains of CD28, CD27, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

[0185] (Intracellular signal transduction domain)

[0186] In some embodiments, intracellular signaling domains may include primary intracellular signaling domains. Example primary intracellular signaling domains include those derived from molecules responsible for primary or antigen-dependent stimulation. In one embodiment, intracellular signaling domains may include co-stimulatory intracellular domains. Example co-stimulatory intracellular signaling domains include those derived from molecules responsible for co-stimulatory signals or antigen-independent stimulation. For example, in the case of CAR-T, the primary intracellular signaling domain may contain a cytoplasmic sequence of a T cell receptor, and the co-stimulatory intracellular signaling domain may contain a cytoplasmic sequence from a co-receptor or co-stimulatory molecule.

[0187] In some implementations, the primary intracellular signaling domain may contain a signaling motif, referred to as an immune receptor tyrosine-based activation motif or ITAM. Examples of primary cytoplasmic signaling sequences containing ITAMs include, but are not limited to, those derived from CD3-ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d, as well as DAP10 and DAP12.

[0188] In this invention, “ζ” or alternatively “ζ chain”, “CD3-ζ”, “TCR-ζ” or “CD3 zeta” is defined as a protein provided in GenBank No. BAG36664.1, or an equivalent residue from a non-human species (e.g., mouse, rabbit, primate, rodent, monkey, ape, etc.), and “ζ-stimulatory domain” or alternatively “CD3-ζ-stimulatory domain” or “TCR-ζ-stimulatory domain” is defined as an amino acid residue from the cytoplasmic domain of the ζ chain sufficient to functionally transmit the initial signal necessary for T cell activation.

[0189] In this invention, "co-stimulatory molecules" refer to homologous binding partners on T cells that specifically bind to co-stimulatory ligands, thereby mediating co-stimulatory responses of T cells, such as, but not limited to, proliferation. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands required for an effective immune response. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, as well as OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), and 4-1BB (CD137).

[0190] The intracellular signal transduction domain of a costimulatory molecule can be the intracellular portion of that molecule. Costimulatory molecules can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signal transduction lymphocyte-activating molecules (SLAM proteins), and activated NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, MyD88, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and ligands that specifically bind to CD83, etc.

[0191] In some specific embodiments of the present invention, the chimeric antigen receptor comprises a sequence as shown in any one of SEQ ID NO:45-49, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NO:45-49.

[0192] <Isolated Polynucleotides>

[0193] This invention provides an isolated polynucleotide that encodes the antibody or antigen fragment targeting CD33 described above.

[0194] In some implementations, the polynucleotide can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded, preferably double-stranded. The DNA can be a coding strand or a non-coding strand. Unless otherwise specified, a particular polynucleotide sequence also implicitly encompasses variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, complementary sequences, and explicitly stated sequences.

[0195] <Expression cassette, recombinant vector, host cell>

[0196] Based on the polynucleotides obtained in this invention, an expression cassette comprising the polynucleotides described in this invention is provided.

[0197] In other embodiments, the present invention provides recombinant vectors comprising polynucleotides or expression cassettes as described above.

[0198] In other embodiments, the present invention provides a host cell comprising a polynucleotide, expression cassette, or recombinant vector as described herein.

[0199] In some specific implementations, the host cell is a bacterium; for example, the host cell is Escherichia coli or Bacillus subtilis, but is not limited thereto.

[0200] In other specific embodiments, the host cell is a yeast (Saccharomyces), exemplarily Pichia Pastoris or Saccharomyces cerevisiae, but not limited to these.

[0201] In other specific embodiments, the host cell is a mammalian cell, exemplarily a mammalian cell line such as HEK293T cells, HEK293F cells, HEK293 cells, CHO cells, but not limited thereto.

[0202] <Recombinant Immune Cells>

[0203] The present invention provides a recombinant immune cell containing the chimeric antigen receptor described above.

[0204] The recombinant immune cells described in this invention can bind to cells expressing CD33 and have a specific killing effect on cells expressing CD33.

[0205] In some specific implementations, the CD33-expressing cells include the CD33-expressing Molm13 cell line.

[0206] In some implementations, the recombinant immune cells are immune cells derived from mammals.

[0207] The mammals mentioned include, but are not limited to, primates (such as humans and monkeys), cattle, sheep, goats, alpacas, horses, dogs, cats, rabbits, rats, mice, etc.

[0208] In this invention, there are no particular restrictions on the types of immune cells. In some embodiments, the immune cells are selected from one or more of T cells, B cells, NK cells, mast cells, and tumor-infiltrating lymphocytes. In some preferred embodiments, the immune cells are selected from T cells or NK cells. In some specific embodiments, the T cells are selected from CD4+ cells. + CD8 + T cells, CD8 + T cells, CD4 + T cells, effector T cells, suppressor T cells, primitive T cells, memory T cells, γ-δ T cells, α-β T cells, CD4+- CD8 - One or more of double-negative T cells or NKT cells. In some preferred embodiments, the T cells are CD8+ cells. + T cells.

[0209] In some specific implementations, the recombinant immune cells are recombinant T cells.

[0210] <Composition>

[0211] The present invention also provides a composition comprising at least one of the above-described CD33-targeting antibody or its antigen-binding fragment, a polynucleotide, a vector, a recombinant cell, a chimeric antigen receptor, and a recombinant immune cell.

[0212] In some alternative embodiments, the composition comprises a pharmaceutical composition.

[0213] Furthermore, the pharmaceutical composition may also include pharmaceutically acceptable carriers and / or excipients. In some embodiments, the pharmaceutically acceptable carriers and / or excipients are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995), and these substances are used as needed to aid in the stability of the formulation or to help improve the bioavailability of the active substance or to produce an acceptable taste or odor when taken orally. The pharmaceutical composition thus formulated can be administered as needed in any suitable manner known to those skilled in the art.

[0214] In some embodiments, the pharmaceutical composition can be prepared into any pharmaceutically conventional dosage form.

[0215] <Reagents, kits, and methods for detection, diagnosis, prevention, or treatment>

[0216] This invention provides reagents or kits comprising at least one of the following: an antibody targeting CD33 as described above, or an antigen-binding fragment thereof, a polynucleotide, a vector, recombinant cells, a chimeric antigen receptor, recombinant immune cells, and a composition. The reagents or kits can be used for the detection and / or diagnosis of CD33. The reagents or kits can also be used for the prevention or treatment of CD33-positive tumors, wherein CD33 positivity includes elevated levels of CD33 at the nucleic acid or protein level.

[0217] In other embodiments of the present invention, a method for detecting CD33 protein in a sample is provided, comprising:

[0218] (a) Contact the sample with an antibody or antigen-binding fragment thereof that targets CD33 as described above;

[0219] (b) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of CD33 protein in the sample.

[0220] In some alternative implementations, the method may be a method for detecting CD33 protein in an in vitro non-diagnostic sample, correspondingly, the above step (a) is performed in vitro.

[0221] In other embodiments of the invention, a kit for the prevention or treatment of tumors is provided, comprising an antibody or antigen-binding fragment thereof targeting CD33 as described above, or a pharmaceutical composition as described above.

[0222] In other embodiments of the invention, a method for preventing or treating tumors in a subject is provided, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof targeting CD33 as described above, a pharmaceutical composition as described above, or a kit for preventing or treating tumors as described above, wherein the tumor is CD33 positive; the CD33 positivity includes having elevated levels of CD33 at the nucleic acid or protein level.

[0223] In some preferred embodiments, the tumor is cancer, specifically myeloid leukemia.

[0224] <Multispecific antibodies>

[0225] This invention provides a multispecific antibody comprising the CD33-targeting antibody or its antigen-binding fragment as described above. In some embodiments, the multispecific antibody further comprises an antigen-binding fragment targeting other antigens, such as an anti-CD3 domain, which can simultaneously target both the cell surface antigen CD33 and the T cell surface trigger molecule CD3, thereby activating T cells and leading to the killing of target cells. The anti-CD3 domain can be a single-chain antibody or an antibody Fab fragment, etc.

[0226] Antibody-Conjugates

[0227] In some embodiments, the present invention provides an antibody-drug conjugate comprising the aforementioned CD33-targeting antibody or its antigen-binding fragment. In some embodiments, the CD33-targeting antibody or its antigen-binding fragment may be conjugated with enzyme labeling, fluorescent labeling, or biotin labeling to obtain the antibody-drug conjugate. In other exemplary embodiments, the antibody-drug conjugate further comprises a cytotoxic drug.

[0228] In this invention, the term "cytotoxic drug" generally refers to a toxic drug that possesses chemical molecules within tumor cells that strongly disrupt their normal growth. Cytotoxic drugs can kill tumor cells at sufficiently high concentrations.

[0229] The term "cytotoxic drug" may include toxins such as small molecule toxins or enzyme-active toxins of bacterial, fungal, plant or animal origin, radioactive isotopes (e.g., radioactive isotopes of At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32 or Lu), toxic drugs, chemotherapeutic drugs, antibiotics or ribolysins, or derivatives thereof.

[0230] Optionally, the cytotoxic drug is a microtubule inhibitor and a DNA topoisomerase inhibitor or other cytotoxic compounds.

[0231] In some embodiments, the cytotoxic drug is MMAE.

[0232] Example

[0233] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific embodiments (although illustrating specific implementations of the invention) are given for illustrative purposes only, as various changes and modifications made within the spirit and scope of the invention will become apparent to those skilled in the art upon reading this detailed description.

[0234] Unless otherwise specified, the experimental techniques and methods used in this embodiment are conventional techniques and methods. For example, experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in the embodiments can be obtained through legitimate commercial channels.

[0235] Example 1. Screening of mouse monoclonal antibodies

[0236] 1. Preparation of human CD33 antigen

[0237] The extracellular sequence information of human CD33 was retrieved from the UniProt database (UniProt Accession#P20138). The sequence from Asp (position 18) to His (position 259) was selected, and a His tag was added to the C-terminus. Codon optimization was performed according to human codon preferences. After gene synthesis, the gene was subcloned into the eukaryotic expression vector pcDNA3.4. After Sanger sequencing verification, plasmid extraction was performed for later use. The constructed eukaryotic protein expression vector was transiently transfected into 293F cells, and the protein expression supernatant was collected and purified using a nickel column. SDS-PAGE was performed to detect protein purity, and Nanodrop 2000 was used to determine protein concentration. The purified protein was used for immunoassay and antibody screening.

[0238] 2. Construction of CHO-K1-CD33 cell line

[0239] Based on the full-length amino acid sequence information of human CD33 in the UniProt database, the gene was synthesized and subcloned into the Lenti-CMV-puro lentiviral expression vector to construct the Lenti-CMV-CD33-puro overexpression vector. After lentiviral packaging, 100 μL of lentiviral material was added to CHO-K1 cells, and after 48 hours, 8 μg / ml puromycin was added for selection of CHO-K1-CD33 cell lines. After cell line selection, the expression of human CD33 on the cell membrane was confirmed by FACS using a positive antibody.

[0240] 3. Mouse immunization

[0241] All mice were housed in a barrier system, fed with sterilized pelleted feed and autoclaved drinking water. Five Balb / c mice (SPF grade) were ear-tagged and immunized subcutaneously at multiple sites with Freund's adjuvant emulsion at a dose of 100 μg / mouse. A total of three immunizations were administered (the first immunization used complete Freund's adjuvant; all subsequent immunizations used incomplete Freund's adjuvant), with immunization intervals of 14 days. Peripheral blood was collected via the tail vein after immunization, and serum was separated. Immunoantibodies were measured using FACS.

[0242] 4. Hybridoma fusion and monoclonal screening

[0243] 1) After immunizing mice with good immune titers for 3 days, obtain the mouse spleen under sterile conditions, prepare B cell single cell suspension, and mix it with SP2 / 0 myeloma cells at a ratio of 1:1. Use BTX cell electrofusion instrument to perform cell fusion.

[0244] 2) After electrofusion, all cells were immediately suspended in complete culture medium (DMEM, 20% FBS and HAT) and seeded into 96-well plates.

[0245] 3) Approximately 10 days after fusion, the medium was changed to HT medium. After two days of culture, the supernatant was collected for ELISA to detect the binding to the target antigen human CD33. At the same time, FACS was used to detect its binding to CHO-K1-CD33 cells. After each round of subcloning, the same steps were followed for ELISA and FACS identification until a single clone was formed.

[0246] 5. Hybridoma sequencing

[0247] Take 1×10 6 The hybridoma cells obtained from the final screening were lysed using Trizol, and total RNA was extracted from the hybridoma cells according to standard methods. This RNA was then reverse transcribed into cDNA, and the variable regions of the heavy and light chains of the antibody were amplified using PCR with hybridoma sequencing primers. TA cloning was then performed, and the PCR-obtained fragments were subcloned into a T vector. Clones were then selected for sequencing. The final antibody sequences were analyzed.

[0248] 6. Hybridoma sequencing results

[0249] Sequencing was performed on positive hybridoma monoclonal cells to obtain the heavy and light chain variable region sequence information of the positive clones, as shown below, where the underlined part represents the CDR region of the antibody.

[0250] Clone 2VH (SEQ ID NO:1)

[0251] QLQQSGAELVGPGTSVKVSCKAS GYAFTNYL IEWVKQRPGQGLEWIGV INPGSGDT NYNEKFKDKA

[0252] TLTADKSSSTAYMQLSSLASDDSAVYFC ARSPNYYGSSYFDY WGQGTTLTVSS

[0253] CDR1: GYAFTNYL (SEQ ID NO:2)

[0254] CDR2: INPGSGDT (SEQ ID NO:3)

[0255] CDR3:ARSPNYYGSSYFDY(SEQ ID NO:4)

[0256] Clone 2VL (SEQ ID NO:5)

[0257] DIVMTQTPKFLLVSAGDRVSITCKAS QNVNYYVAWYQQKPGQSPKLLIY YAS SRYAGVPDRFTGSGY

[0258] GTDFTFTISTVQAEDLAVYFC QQDYNSPYT FGGGTKLELK

[0259] CDR1: QNVNYY (SEQ ID NO:6)

[0260] CDR2: YAS

[0261] CDR3: QQDYNSPYT (SEQ ID NO:7)

[0262]

[0263] CDR1: QDINKY (SEQ ID NO:13)

[0264] CDR2: YTS

[0265] CDR3: LHYENLLT (SEQ ID NO:32)

[0266] Example 2. Antibody humanization design and affinity detection

[0267] Humanization was designed based on the obtained amino acid sequences of the mouse antibody heavy and light chains, retaining the original antibody's CDR region sequence. Based on germline alignment and antibody structure simulation results, different human antibody templates were selected for the heavy and light chains, and back mutations were performed in the humanized frame region to design candidate humanized antibody sequences. The heavy and light chains of the humanized antibodies were synthesized separately. The heavy chain was subcloned into the pcDNA3.4-hIgG1 expression vector to construct the antibody heavy chain expression vector, and the light chain was subcloned into the pcDNA3.4-hIgKc expression vector to construct the antibody light chain expression vector. After sequencing verification, the vectors were transiently transfected into 293F cells. After collecting the culture supernatant, the recombinant humanized antibody was purified using Protein A, and FACS was used to monitor the binding of Clone2-HM, Clone3-HM, Clone6-HM, and Clone7-HM to recombinant CHO-CD33 cells overexpressing the target gene CD33.

[0268] The amino acid sequence of the hIgG1 heavy chain constant region (SEQ ID NO:43):

[0269] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0270] hIgKc light chain constant region amino acid sequence (SEQ ID NO:44):

[0271] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0272] The heavy and light chain variable region sequence information of the humanized antibody sequence is as follows, where the underlined part represents the antibody's CDR region.

[0273] Clone2_VH-HM(SEQ ID NO:33)

[0274] QLQLVQSGAEVKKPGASVKVSCKAS GYAFTNYL IEWVRQAPGQGLEWIGV INPGSGDT NYNEKFQ

[0275] GRVTLTADKSISTAYMELSRLRSDDTAVYFC ARSPNYYGSSYFDY WGQGTLVTVSS

[0276] CDR1: GYAFTNYL (SEQ ID NO:2)

[0277] CDR2: INPGSGDT (SEQ ID NO:3)

[0278] CDR3:ARSPNYYGSSYFDY(SEQ ID NO:4)

[0279] >Clone2_VL-HM(SEQ ID NO:34)

[0280] DIVMTQSPDSLAVSLGERATINCKAS QNVNYY VAWYQQKPGQSPKLLIY YAS SRYAGVPDRFTGSG

[0281] YGTDFTLTISSLQAEDVAVYFC QQDYNSPYT FGGGTKLELK

[0282] CDR1:QNVNYY(SEQ ID NO:6)

[0283] CDR2:YAS

[0284] CDR3:QQDYNSPYT(SEQ ID NO:7)

[0285] >Clone3_VH-HM(SEQ ID NO:35)

[0286] QVQLVQSGAEVKKPGASVKVSCKAS GYTFTRYW MYWVRQAPGQGLEWIGE INPSNGQT NYAQKF

[0287] QGRATLTVDKSTSTAYMELSSLRSEDTAVYYC ARWHYGLDY WGQGTTVTVSS

[0288] CDR1:GYTFTRYW(SEQ ID NO:9)

[0289] CDR2:INPSNGQT(SEQ ID NO:10)

[0290] CDR3:ARWHYGLDY(SEQ ID NO:11)

[0291] >Clone3_VL-HM(SEQ ID NO:36)

[0292] DIQMTQSPSSLSASVGDRVTITCKAS QDINKY IAWYQHKPGKGPELLIY YTS TLHPGVPSRFSGSGSG

[0293] RDYTLTISSLQPEDFATYYC LHYDNLLT FGGGTKVEIK

[0294] CDR1: QDINKY (SEQ ID NO:13)

[0295] CDR2:YTS

[0296] CDR3: LHYDNLLT(SEQ ID NO:14)

[0297] >Clone5_VH-HM(SEQ ID NO:37)

[0298] QVTLKESGPTLVKPTQTLTLTCTFS GFSLNTSGLG VGWSRQPPGKALEWLAL IWWDDIK YYSPSLKS

[0299] RLTITKDTSKNQVVLTMTNMDPVDTATYYC ARRGHNNAMDY WGQGTLVTVSS

[0300] CDR1:GFSLNTSGLG(SEQ ID NO:16)

[0301] CDR2: IWWDDIK (SEQ ID NO:17)

[0302] CDR3:ARRGHNNAMDY(SEQ ID NO:18)

[0303] >Clone5_VL-HM(SEQ ID NO:38)

[0304] DIQMTQSPSSLSASVGDRVTITCKAN QDINQY IAWYQQKPGKGPKLLIY YTS LFQPGVPSRFSGSGSG

[0305] RDYTLTISSLQPEDFATYYC LHYGNLLWT FGGGTKVEIK

[0306] CDR1: QDINQY (SEQ ID NO:20)

[0307] CDR2:YTS

[0308] CDR3:LHYGNLLWT(SEQ ID NO:21)

[0309] >Clone6_VL-HM(SEQ ID NO:39)

[0310] DIQMTQSPSSLSASVGDRVTITCQAS QDISNY LNWYQQKPGGAVKLLIY YTS RLHTGVPSRFSGSGS

[0311] GTDYTFTISSLQPEDIATYYC QQGDTLPWT FGGGTKVEIK

[0312] CDR1:QDISNY(SEQ ID NO:27)

[0313] CDR2:YTS

[0314] CDR3:QQGDTLPWT(SEQ ID NO:28)

[0315] >Clone6_VH-HM(SEQ ID NO:40)

[0316] EVKLVESGGGLVQPGGSLRLSCAAS GFTFSNYA MSWVRQAPGKGLEWVAS ISSGGDT YYPDSVKG

[0317] RFTISRDNSKNTLYLQMNSLRAEDTAVYYC VRGEANWDYFDY WGQGTLVTVSS

[0318] CDR1:GFTFSNYA(SEQ ID NO:23)

[0319] CDR2:ISSGGDT(SEQ ID NO:24)

[0320] CDR3:VRGEANWDYFDY(SEQ ID NO:25)

[0321] >Clone7_VH-HM(SEQ ID NO:41)

[0322] QVQLVQSGAEVKKPGASVKVSCKAS GYTFTRYW IYWARQAPGQGLEWIGE IKPSDGRT NYAQKFQ

[0323] GRATLTVDESTSTVYMELSSLRSEDTAVYYC ARWHYGLDY WGQGTTVTVSS

[0324] CDR1:GYTFTRYW(SEQ ID NO:9)

[0325] CDR2: IKPSDGRT (SEQ ID NO:30)

[0326] CDR3:ARWHYGLDY(SEQ ID NO:11)

[0327] Clone7_VL-HM(SEQ ID NO:42)

[0328] DIQMTQSPSSLSASVGDRVTITCKSS QDINKY IAWYQHKPGKGPKLLIR YTS ILQPGVPSRFSGSGSGR

[0329] DYTLTISSLQPEDFATYYC LHYENLLT FGGGTKVEIK

[0330] CDR1: QDINKY (SEQ ID NO:13)

[0331] CDR2: YTS

[0332] CDR3: LHYENLLT (SEQ ID NO:32)

[0333] The prepared humanized antibodies were subjected to affinity testing, and the results are shown in Table 1.

[0334] Table 1

[0335]

[0336] Example 3. Killing of target cells by T cells expressing candidate antibody chimeric antigen receptor

[0337] 1. CAR-T cell preparation

[0338] Based on the results of humanization testing, Clone2HM, Clone3HM, Clone5HM, Clone6HM, and Clone7HM were selected to construct chimeric antigen receptor lentiviral expression vectors (activation signals are 4-1BB and CD3Zeta), respectively. CAR lentiviral expression vectors were then constructed, and after preparing recombinant lentiviruses, they were used to infect primary T cells to prepare CAR-T cells for pharmacodynamic evaluation.

[0339] >Clone2HM CAR amino acid sequence (SEQ ID NO:45, the underlined part is the CD8 Hinge region sequence, the double underlined part is the CD8 transmembrane region sequence, the bolded part is the 4-1BB intracellular signal domain sequence, the italicized part is the CD3Zeta intracellular signal domain, and the remaining part is the antibody light chain variable region and heavy chain variable region sequence)

[0340]

[0341] >Clone3HM CAR amino acid sequence (SEQ ID NO:46, the underlined part is the CD8 Hinge region sequence, the double underlined part is the CD8 transmembrane region sequence, the bolded part is the 4-1BB intracellular signal domain sequence, the italicized part is the CD3Zeta intracellular signal domain, and the remaining part is the antibody light chain variable region and heavy chain variable region sequence)

[0342]

[0343] >Clone5HM CAR amino acid sequence (SEQ ID NO:47, the underlined part is the CD8 Hinge region sequence, the double underlined part is the CD8 transmembrane region sequence, the bolded part is the 4-1BB intracellular signal domain sequence, the italicized part is the CD3Zeta intracellular signal domain sequence, and the remaining part is the antibody light chain variable region and heavy chain variable region sequence)

[0344]

[0345] >Clone6HM CAR amino acid sequence (SEQ ID NO:48, the underlined part is the CD8 Hinge region sequence, the double underlined part is the CD8 transmembrane region sequence, the bolded part is the 4-1BB intracellular signal domain sequence, the italicized part is the CD3Zeta intracellular signal domain, and the remaining part is the antibody light chain variable region and heavy chain variable region sequence)

[0346]

[0347] >Clone7HM CAR amino acid sequence (SEQ ID NO:49, the underlined part is the CD8 Hinge region sequence, the double underlined part is the CD8 transmembrane region sequence, the bolded part is the 4-1BB intracellular signal domain sequence, the italicized part is the CD3Zeta intracellular signal domain, and the remaining part is the antibody light chain variable region and heavy chain variable region sequence)

[0348]

[0349] 2. Lysis of target cells by CAR-T cells

[0350] 1) Adjust the Molm13-Luciferase target cells to the logarithmic growth phase, resuspend the target cells in complete culture medium, and adjust the cell density to 2 × 10⁶ cells / year. 5To obtain a target cell count of 100 cells / mL, take a new 96-well plate and seed it with target cells at a rate of 100 cells per well. Add 100 μL of sterile water to each of the unused wells around the perimeter of the 96-well plate to prevent water evaporation from the central wells. Place the plate in a 5% CO2, 37°C incubator and incubate overnight.

[0351] 2) Collect the prepared CAR-T cells by centrifugation and resuspend them in 1640 medium with 10% FBS. Remove the 96-well plate from the incubator, completely aspirate the medium from the wells, gently wash the cells once with sterile PBS, and then add CAR-T cells according to different CAR-T cell to target cell ratios, bringing the final volume to 200 μL / well. Maxilysis involves seeding the same number of target cells but without adding CAR-T cells. Place the plate in a 5% CO2, 37°C incubator and incubate for 18 hours.

[0352] 3) After culture, the well plates were removed from the incubator, and the luciferase activity was measured to reflect the lytic ability of recombinant CAR-T cells to target cells. Simultaneously, the supernatant from the co-cultured medium was collected, and ELISA was used to detect changes in cytokine secretion and expression levels after CAR-T cell activation.

[0353] 4) Formula for calculating the percentage of target cell lysis:

[0354]

[0355] Among them, RLU 样本 RLU represents the fluorescence intensity of samples co-cultured with different CAR-T cells and target cells. 最大值 This indicates the fluorescence intensity only for samples cultured from target cells.

[0356] result:

[0357] Using Molm13-Luciferase as the target cell and CAR-T cells expressing different humanized antibodies as effector cells, according to... Figure 3 An in vitro co-culture system was established based on the effector-target ratio. The results showed that, compared with the control T cells, the CAR-T cells constructed with the candidate antibody in this invention had a strong killing effect on Molm13-Luciferase cells; and the killing effect was consistent with that of CAR-T cells constructed based on the positive control antibody Gentuzumab.

[0358] Using Molm13-Luc as the target cell and the prepared CAR-T cells as the effector cells, according to... Figure 4In vitro co-culture systems were established using effector-target ratios of 1:1, 2.5:1, 5:1, and 10:1. The results showed that, compared with blank T cells, CAR-T cells stimulated by Molm13-Luciferase cells exhibited significantly enhanced IFN-γ secretion, consistent with CAR-T cells constructed based on the positive control antibody Gemtuzumab.

[0359] Example 4. CAR in vivo efficacy verification

[0360] 1. Animal modeling and grouping

[0361] Fifteen 6-8 week old NSG mice (weighing 18-22g) were acclimatized for one week and then injected via tail vein with the CD33-positive tumor cell line Molm13-luciferase. Each mouse was injected with 3.3 × 10⁻⁶ cells. 5 Seven days after tumor cell inoculation, mice were randomly divided into five groups (PBS, UTD, Clone2HM, Clone6HM, and CD33 control group) based on body weight. CAR-T cells or control T cells were infused via tail vein (D0 / D1 administration), with each mouse receiving 1.5 × 10⁻⁶ tumor cells. 7 One positive CAR-T cell was observed. The animal's condition was closely monitored, and the first in vivo imaging was performed on day 7, followed by imaging every 7 days thereafter, for a total of 4 imaging sessions.

[0362] 2. Experimental Observation

[0363] Following tail vein inoculation with Molm-13-luciferase-expressing cells, CAR-T cells were directly reinfused 7 days later. After grouping the mice, CAR-T cells were infused via tail vein, and in vivo imaging was performed every 7 days for a total of 4 imaging sessions.

[0364] Based on imaging detection, the results are as follows: Figure 5 As shown, CAR-T cells constructed based on the humanized antibody of the present invention can significantly inhibit the growth of tumor cell lines in mice.

[0365] Example 5. Killing effect of candidate antibody on target cells after conjugation with toxin

[0366] 1. Antibody conjugation

[0367] Six equivalents of TCEP were added to candidate antibodies Clone2-HM, Clone3-HM, Clone5-HM, Clone6-HM, and Clone7-HM, respectively, and the mixture was reduced at 25°C for 6 hours. Then, ten equivalents of Mal-PEG8-Val-Cit-PAB-MMAE were added for further conjugation for 4 hours. After conjugation, unreacted Mal-PEG8-VC-PAB-MMAE was removed using Protein A-binding antibody. The antibody was eluted from the affinity chromatography column with glycine at pH 3.0, and then replaced with PBS buffer. The conjugated products were analyzed using HIC-HPLC, and the average DAR value was calculated. The positive control antibody Gemtuzumab was conjugated to MMAE toxin using the same protocol.

[0368] 2. In vitro killing effect of candidate antibodies on target cells after conjugation with MMAE

[0369] 1) Take Molm13-Luciferase cells in the logarithmic growth phase and adjust the cell density to 2 × 10⁶ cells / year using complete culture medium. 5 per mL.

[0370] 2) Add 100 μL of sterile water to the wells around the perimeter of the 96-well plate; add 100 μL of cell suspension to the remaining wells in sequence, and incubate in a 5% CO2, 37°C incubator for 24 h.

[0371] 3) After sterilizing the candidate antibody and positive antibody conjugate by filtration through a 0.22 μm filter membrane, the antibody concentration was adjusted to 20 μg / mL using complete culture medium.

[0372] 4) Take 240 μL of the initial gradient concentration stock solution (antibody concentration of 20 μg / mL) and add it to 480 μL of culture medium. Perform a 3-fold serial dilution for a total of 9 dilutions. Add 100 μL of antibody diluent to each well.

[0373] 5) 120 h after drug administration, add 100 μL of cell viability assay kit substrate to each cell well, pipette 20 times, let stand for 10 min, transfer 100 μL of liquid from each well to a white non-transparent 96-well plate, and read the luminescence value using a Tecan M1000Pro.

[0374] Candidate antibodies and positive control antibodies conjugated with toxins effectively inhibited the proliferation of tumor cells Molm13-Luciferase; and all candidate antibodies conjugated with toxins showed stronger cytotoxic activity against target cells than the positive control antibody Gemtuzumab. Figure 6 ).

[0375] 3. Killing of target cells in vivo after candidate antibody conjugation with MMAE

[0376] 1) Resuscitate Molm-13-Luciferase cells in liquid nitrogen, adjust to the logarithmic growth phase, and then adjust the cell density to 5 x 10⁻⁶ cells / year using physiological saline. 6 / mL;

[0377] 2) Take NSG mice and inoculate them with 5 x 10 Molm13-Luciferase cells via the tail vein. 5 / mice, inoculation volume is 100μL;

[0378] 3) On the 7th day after tumor grafting, mice were injected subcutaneously into the peritoneum with 200 μL of 10 mg / mL D-fluorescein potassium for the first in vivo imaging experiment. On the same day, mice were infused with different antibody-drug conjugates (Gemtuzumab-MMAE, Clone3-HM-MMAE) at a dose of 5 mg / kg, while control mice were injected with the same volume of physiological saline via the tail vein.

[0379] 4) In vivo imaging was performed every 7 days thereafter to monitor the proliferation of tumor cells in the mice.

[0380] The candidate antibody Clone3-HM and the positive control antibody, after being conjugated with the toxin, effectively inhibited the proliferation of Molm13-Luciferase tumor cells in mice, and their cytotoxic activity against target cells was stronger than that of the positive control antibody Gemtuzumab. Figure 7 Mice survived longer. Figure 8 The mice did not experience a significant decrease in body weight throughout the administration period. Figure 9 ).

Claims

1. An antibody or antigen-binding fragment thereof targeting CD33, comprising a heavy chain variable region and a light chain variable region, wherein, the light chain variable region comprises: an LCDR1 as set forth in SEQ ID NO: 6, SEQ ID NO: 13, SEQ ID NO: 20, or SEQ ID NO: 27; an LCDR2 as set forth in YAS or YTS; an LCDR3 as set forth in SEQ ID NO: 7, SEQ ID NO: 14, SEQ ID NO: 21, SEQ ID NO: 28, or SEQ ID NO: 32; the heavy chain variable region comprises: an HCDR1 as set forth in SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 16, or SEQ ID NO: 23; an HCDR2 as set forth in SEQ ID NO: 3, SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 24, or SEQ ID NO: 30; an HCDR3 as set forth in SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 18, or SEQ ID NO:

25.

2. The antibody or antigen-binding fragment thereof targeting CD33 of claim 1, wherein, the light chain variable region comprises an LCDR1, an LCDR2, and an LCDR3 selected from any one of the following (al) to (a5): (al) an LCDR1 as set forth in SEQ ID NO: 6, an LCDR2 as set forth in YAS, or an LCDR3 as set forth in SEQ ID NO: 7; (a2) an LCDR1 as set forth in SEQ ID NO: 13, an LCDR2 as set forth in YTS, or an LCDR3 as set forth in SEQ ID NO: 14; (a3) an LCDR1 as set forth in SEQ ID NO: 20, an LCDR2 as set forth in YTS, or an LCDR3 as set forth in SEQ ID NO: 21; (a4) an LCDR1 as set forth in SEQ ID NO: 27, an LCDR2 as set forth in YTS, or an LCDR3 as set forth in SEQ ID NO: 28; (a5) an LCDR1 as set forth in SEQ ID NO: 13, an LCDR2 as set forth in YTS, or an LCDR3 as set forth in SEQ ID NO: 32; the heavy chain variable region comprises an HCDR1, an HCDR2, and an HCDR3 selected from any one of the following (bl) to (b5): (bl) an HCDR1 as set forth in SEQ ID NO: 2, an HCDR2 as set forth in SEQ ID NO: 3, or an HCDR3 as set forth in SEQ ID NO: 4; (b2) an HCDR1 as set forth in SEQ ID NO: 9, an HCDR2 as set forth in SEQ ID NO: 10, or an HCDR3 as set forth in SEQ ID NO: 11; (b3) an HCDR1 as set forth in SEQ ID NO: 16, an HCDR2 as set forth in SEQ ID NO: 17, or an HCDR3 as set forth in SEQ ID NO: 18; (b4) HCDR1 as depicted in SEQ ID NO: 23, HCDR2 as depicted in SEQ ID NO: 24 or HCDR3 as depicted in SEQ ID NO: 25; (b5) HCDR1 as depicted in SEQ ID NO: 9, HCDR2 as depicted in SEQ ID NO: 30 or HCDR3 as depicted in SEQ ID NO:

11.

3. The antibody or antigen-binding fragment thereof targeting CD33 of claim 1 or 2, wherein, The antibody or antigen-binding fragment thereof targeting CD33 comprises a murine antibody or fragment thereof, a chimeric antibody or fragment thereof, a humanized antibody or fragment thereof and / or a fully human antibody or fragment thereof.

4. The antibody or antigen-binding fragment thereof targeting CD33 according to claim 3, wherein, the light chain variable region of the murine antibody or fragment thereof comprises a sequence as depicted in any one of SEQ ID NOs: 5, 12, 19, 26 and 31, or a sequence having at least 97%, 98% or 99% identity to any one of SEQ ID NOs: 5, 12, 19, 26 and 31; the heavy chain variable region of the murine antibody or fragment thereof comprises a sequence as depicted in any one of SEQ ID NOs: 1, 8, 15, 22 and 29, or a sequence having at least 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NOs: 1, 8, 15, 22 and 29.

5. The antibody or antigen-binding fragment thereof targeting CD33 according to claim 3, wherein, the light chain variable region of the humanized antibody or fragment thereof comprises a sequence as depicted in any one of SEQ ID NOs: 34, 36, 38, 39 and 42, or a sequence having at least 97%, 98% or 99% identity to any one of SEQ ID NOs: 34, 36, 38, 39 and 42; the heavy chain variable region of the humanized antibody or fragment thereof comprises a sequence as depicted in any one of SEQ ID NOs: 33, 35, 37, 40 and 41, or a sequence having at least 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NOs: 33, 35, 37, 40 and 41.

6. The antibody or antigen-binding fragment thereof targeting CD33 according to claim 5, which comprises a heavy chain constant region of a human IgGl or variant thereof, a human IgG2 or variant thereof, a human IgG3 or variant thereof or a human IgG4 or variant thereof; and / or, which comprises a light chain constant region derived from a human kappa chain, a lambda chain or a variant thereof.

7. The antibody or antigen-binding fragment thereof targeting CD33 of any one of claims 1-6, wherein, The antigen-binding fragment is selected from at least one of F(ab)2, Fab’, Fab, Fv, scFv, a multispecific antibody and a single chain antibody.

8. A chimeric antigen receptor, comprising: (A) an extracellular domain that specifically binds to CD33; (B) a transmembrane domain; (C) an intracellular signaling domain; Preferably, the extracellular domain comprises the antibody or antigen-binding fragment thereof targeting CD33 according to any one of claims 1 to 7; More preferably, the chimeric antigen receptor comprises a sequence as set forth in any one of SEQ ID NOs: 45-49, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 45-49.

9. An isolated polynucleotide, wherein, The polynucleotide encodes the antibody or antigen-binding fragment thereof targeting CD33 of any one of claims 1-7 or the chimeric antigen receptor of claim 8.

10. An expression cassette, wherein, The expression cassette comprises the polynucleotide of claim 9.

11. An expression vector, wherein, The expression vector contains the polynucleotide of claim 9 or the expression cassette of claim 10.

12. A host cell, wherein, The host cell comprises the polynucleotide of claim 9, the expression cassette of claim 10, or the expression vector of claim 11.

13. A recombinant immune cell, wherein, The recombinant immune cell comprises the chimeric antigen receptor of claim 8.

14. The recombinant immune cell of claim 13, wherein, The immune cell is an immune cell derived from a mammal; Optionally, the immune cell is selected from one or more of a T cell, a B cell, an NK cell, a macrophage, a tumor infiltrating lymphocyte; Preferably, the immune cell is selected from a T cell, an NK cell, or a macrophage. More preferably, the T cells are selected from CD4 + CD8 + T cells, CD8 + T cells, CD4 + T cells, effector T cells, suppressor T cells, naive T cells, memory T cells, gamma-delta T cells, alpha-beta T cells, CD4 - CD8 - one or more of double negative T cells and NKT cells.

15. Use of the antibody or antigen-binding fragment thereof targeting CD33 of any one of claims 1-7, the chimeric antigen receptor of claim 8, the polynucleotide of claim 9, the expression cassette of claim 10, the expression vector of claim 11, the host cell of claim 12, or the recombinant immune cell of claim 13 or 14 in the manufacture of a medicament for treating a tumor. Optionally, the tumor is CD33 positive. Preferably, the tumor is a cancer.

16. A multispecific antibody, wherein The multispecific antibody comprises the antibody or antigen-binding fragment thereof targeting CD33 of any one of claims 1-7.

17. A composition comprising at least one of the antibody or antigen-binding fragment thereof targeting CD33 of any one of claims 1-7, the chimeric antigen receptor of claim 8, the polynucleotide of claim 9, the expression cassette of claim 10, the expression vector of claim 11, the host cell of claim 12, and the recombinant immune cell of claim 13 or 14. Optionally, the composition comprises a pharmaceutical composition. Optionally, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and / or excipient.

18. A detection or diagnostic reagent or kit comprising the antibody or antigen-binding fragment thereof targeting CD33 of any one of claims 1-7.

19. An in vitro non-diagnostic method of detecting a CD33 protein in a sample, comprising the steps of: (a) in vitro, contacting the sample with the antibody or antigen-binding fragment thereof targeting CD33 of any one of claims 1-7; (b) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of the CD33 protein in the sample.

20. An antibody conjugate, wherein, The antibody conjugate comprises the antibody or antigen binding fragment thereof targeting CD33 as claimed in any one of claims 1 to 7; Optionally, the antibody conjugate comprises a cytotoxic drug; Optionally, the cytotoxic drug is monomethyl auristatin (MMAE).

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