Humanized antibody targeting human CD33 and use thereof

Abstract

Provided are a monoclonal antibody targeting human CD33 and a humanized antibody thereof, and also provided are a composition comprising the antibody, a multispecific antibody, a chimeric antigen receptor, and an antibody conjugate, which facilitate prediction, diagnosis and treatment monitoring of related diseases such as myeloid leukemia.

Description

Humanized antibodies targeting human CD33 and uses thereof

[0001] The present disclosure claims priority to the Chinese patent application No. 202411280813.0, filed on September 12, 2024, and entitled "Humanized antibodies targeting human CD33 and uses thereof", the entire content of which is incorporated herein by reference. TECHNICAL FIELD

[0002] The present disclosure belongs to the technical field of biological medicine, and relates to humanized antibodies targeting human CD33 and uses thereof, further relates to humanized antibodies targeting human CD33 and uses thereof in myeloid leukemia, in particular to the construction of CAR-T cell drugs expressing humanized CD33 monoclonal antibodies and the application of antibody conjugate drugs in the killing of Molm13-Luciferase cells. BACKGROUND

[0003] CD33 is a single transmembrane protein highly expressed in myeloid cells; it is highly expressed in acute myeloid leukemia progenitor cells, but not expressed in normal stem cells. According to existing research reports, CD33 is mainly distributed in myeloblasts, monocytes and myelocytes, and its expression in non-hematopoietic tissues is very limited, but the expression level in myeloid leukemia cells is relatively high. During the differentiation of multi-functional hematopoietic stem cells, the expression level of CD34 decreases, and CD33 appears [1] , which makes this molecule an ideal target for myeloid leukemia treatment. Gemtuzumab ozogamicin (Gemtuzumab, disclosed in patent US7727968B2) is a humanized monoclonal IgG4 antibody targeting human CD33, which is coupled with a cytotoxic calicheamicin derivative. It binds to CD33-expressing leukemia blast cells and is internalized when the linker is hydrolyzed, releasing the toxin to bind to DNA, causing site-specific double-strand breaks, thereby causing cell death. Chimeric antigen receptor T cells based on this antibody have a significant killing effect on CD33-expressing myeloid leukemia cells [2] . In addition, antibodies targeting human CD33 are combined with agonist antibodies targeting human CD3 to form bispecific antibody fusion proteins, which can also produce significant killing of myeloid leukemia cells after bridging T cells [3] . The present disclosure immunizes mice to obtain monoclonal antibodies that specifically bind to human CD33, and further humanizes them; chimeric antigen receptor T cells based on humanized antibodies and conjugated with MMAE toxins can produce significant killing of tumor cells.

[0004] REFERENCES

[0005] 1. Andrews, R. G., J. W. Singer, and I. D. Bernstein, 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.

[0006] 2. Kenderian, S. S., et al., CD33-specific chimeric antigen receptor T cells exhibit potent preclinical activity against human acute myeloid leukemia. Leukemia, 2015. 29(8): p. 1637-47.

[0007] 3. Hoseini, S. S., et al., T cell engaging bispecific antibodies targeting CD33 IgV and IgC domains for the treatment of acute myeloid leukemia. J Immunother Cancer, 2021. 9(5). SUMMARY

[0008] Problems to be solved by the invention

[0009] Based on the problems existing in the prior art, the present disclosure screened a monoclonal antibody that can specifically bind to CD33, further constructed a CAR-T cell expressing a humanized CD33 monoclonal antibody, evaluated its killing effect on Molm13-Luciferase cells, and also prepared an antibody conjugate and evaluated its effect on target cells in vivo and in vitro.

[0010] Solution to the problem

[0011] The first aspect of the present disclosure provides an antibody or antigen binding fragment thereof targeting CD33, comprising a heavy chain variable region and a light chain variable region, wherein,

[0012] the light chain variable region comprises:

[0013] LCDR1 as set forth in SEQ ID NO: 6, SEQ ID NO: 13, SEQ ID NO: 20, or SEQ ID NO: 27;

[0014] LCDR2 as set forth in YAS or YTS;

[0015] 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;

[0016] the heavy chain variable region comprises:

[0017] HCDR1 as set forth in SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 16, or SEQ ID NO: 23;

[0018] 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;

[0019] HCDR3 as set forth in SEQ ID NO: 4 SEQ ID NO: 11, SEQ ID NO: 18, or SEQ ID NO: 25.

[0020] In some embodiments, the light chain variable region comprises LCDR1, LCDR2, and LCDR3 selected from any one of (a1) to (a5):

[0021] (a1) LCDR1 as set forth in SEQ ID NO: 6, LCDR2 as set forth in YAS, or LCDR3 as set forth in SEQ ID NO: 7;

[0022] (a2) LCDR1 as set forth in SEQ ID NO: 13, LCDR2 as set forth in YTS, or LCDR3 as set forth in SEQ ID NO: 14;

[0023] (a3) LCDR1 as set forth in SEQ ID NO: 20, LCDR2 as set forth in YTS, or LCDR3 as set forth in SEQ ID NO: 21;

[0024] (a4) LCDR1 as set forth in SEQ ID NO: 27, LCDR2 as set forth in YTS, or LCDR3 as set forth in SEQ ID NO: 28;

[0025] (a5) LCDR1 as shown in SEQ ID NO: 13, LCDR2 as shown in YTS, or LCDR3 as shown in SEQ ID NO: 32;

[0026] the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 selected from any one of the following (b1) to (b5):

[0027] (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;

[0028] (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;

[0029] (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;

[0030] (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;

[0031] (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.

[0032] In some embodiments, 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.

[0033] In some embodiments, the light chain variable region of the murine antibody or fragment thereof comprises a sequence as shown 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.

[0034] the heavy chain variable region of the murine antibody or fragment thereof comprises a sequence as shown 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.

[0035] In some embodiments, the light chain variable region of the humanized antibody or fragment thereof comprises a sequence as set forth 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, or 42;

[0036] the heavy chain variable region of the humanized antibody or fragment thereof comprises a sequence as set forth 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.

[0037] In some embodiments, the antibody or antigen-binding fragment thereof targeting CD33 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.

[0038] and / or,

[0039] comprises a light chain constant region derived from a human kappa chain, lambda chain, or variant thereof.

[0040] In some embodiments, the antigen-binding fragment is selected from at least one of F(ab)2, Fab’, Fab, Fv, scFv, multispecific antibody, and single-chain antibody.

[0041] A second aspect of the present disclosure provides a chimeric antigen receptor comprising:

[0042] (A) an extracellular domain that specifically binds to CD33;

[0043] (B) a transmembrane domain;

[0044] (C) an intracellular signaling domain;

[0045] Preferably, the extracellular domain comprises an antibody or antigen-binding fragment thereof targeting CD33 as described above;

[0046] 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.

[0047] A third aspect of the present disclosure provides an isolated polynucleotide, wherein the polynucleotide encodes the antibody or antigen-binding fragment thereof targeting CD33 as described above or the chimeric antigen receptor as described above.

[0048] A fourth aspect of the present disclosure provides an expression cassette, wherein the expression cassette comprises the polynucleotide as described above.

[0049] A fifth aspect of the present disclosure provides an expression vector, wherein the expression vector contains the polynucleotide as described above or the expression cassette as described above.

[0050] A sixth aspect of the present disclosure provides a host cell, wherein the host cell comprises the polynucleotide as described above, the expression cassette as described above or the expression vector as described above.

[0051] A seventh aspect of the present disclosure provides a recombinant immune cell, wherein the recombinant immune cell comprises the chimeric antigen receptor as described above.

[0052] In some embodiments, the immune cell is a mammalian immune cell;

[0053] 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;

[0054] Preferably, the immune cell is selected from a T cell or an NK cell or a macrophage;

[0055] More preferably, the T cell is selected from one or more of a CD4 + CD8 + T cell, a CD8 + T cell, a CD4 + T cell, an effector T cell, a suppressor T cell, a naive T cell, a memory T cell, a gamma-delta T cell, an alpha-beta T cell, a CD4 - CD8 - a double negative T cell and a NKT cell.

[0056] The present disclosure provides 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 manufacture of a medicament for treating a tumor;

[0057] Optionally, the tumor is CD33 positive;

[0058] Preferably, the tumor is a cancer.

[0059] The present disclosure also provides a multispecific antibody, wherein the multispecific antibody comprises the antibody or antigen-binding fragment thereof targeting CD33 as described above.

[0060] The present disclosure provides a composition comprising at least one 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 and the recombinant immune cell as described above.

[0061] Optionally, the composition comprises a pharmaceutical composition.

[0062] Optionally, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and / or excipient.

[0063] The present disclosure provides a detection or diagnosis reagent or kit comprising the antibody or antigen-binding fragment thereof targeting CD33 as described above.

[0064] The present disclosure provides a method for detecting CD33 protein in a sample in vitro, which comprises the steps of:

[0065] (a) contacting the sample with the antibody or antigen-binding fragment thereof targeting CD33 as described above in vitro;

[0066] (b) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of CD33 protein in the sample.

[0067] The present disclosure provides an antibody conjugate, wherein the antibody conjugate comprises the antibody or antigen-binding fragment thereof targeting CD33 as described above.

[0068] Optionally, the antibody conjugate comprises a cytotoxic drug.

[0069] Optionally, the cytotoxic drug is monomethyl auristatin E (MMAE).

[0070] Effects of the invention

[0071] The antibody targeting CD33 provided by the present disclosure has high affinity and can bind to mammalian cells expressing CD33.

[0072] The antibody targeting CD33, the chimeric antigen receptor and the antibody conjugate provided by the present disclosure have good killing effect on target cells and inhibit the growth of tumors.

[0073] The method for detecting CD33 protein in a sample in vitro provided by the present disclosure, the detection or diagnosis kit comprising the antibody targeting CD33 can also effectively detect CD33 protein. BRIEF DESCRIPTION OF DRAWINGS

[0074] Figure 1 shows the results of FACS detection of binding of candidate clones to target protein.

[0075] Figure 2 shows the results of affinity detection of prepared humanized antibodies.

[0076] Figure 3 shows the results of killing detection of target cells by candidate humanized antibody constructed chimeric antigen receptor T cells.

[0077] Figure 4 shows the results of cytokine secretion detection of CAR-T cells.

[0078] Figure 5 shows the results of in vivo efficacy detection of CAR-T.

[0079] Figure 6 shows the results of killing detection of target cells after antibody conjugated toxin.

[0080] Figure 7 shows the results of killing of tumor cells in mice after antibody conjugated toxin.

[0081] Figure 8 shows the effect on the survival time of mice after antibody conjugated toxin.

[0082] Figure 9 shows the effect on the body weight of mice after antibody conjugated toxin. DETAILED DESCRIPTION

[0083] For the purpose of facilitating the understanding of the present disclosure, certain technical and scientific terms are defined below. Unless otherwise defined herein, all other technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0084] In the present disclosure, the meaning of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0085] In the present disclosure, "optional" or "optionally" means that the event or circumstance described next can or can not occur, and the description includes both the case where the event occurs and the case where the event does not occur.

[0086] In the present disclosure, "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", and the like refer to the specific elements (e.g., features, structures, properties, and / or characteristics) described in relation to the embodiments being included in at least one of the embodiments described herein, and can or can not be present in other embodiments. In addition, it should be understood that the elements can be combined in various embodiments in any suitable manner.

[0087] In the present specification, "preferably", "preferred" do not limit the scope of the claimed disclosure, or imply that certain features are critical, essential, or even important to the structure or function of the claimed disclosure. Instead, these terms merely indicate that the alternative or additional features are / are not used in some embodiments of the disclosure.

[0088] In the present disclosure, if there is no special description, the "comprising" mentioned herein means open or closed. For example, the "comprising" can mean that it can also contain other components not listed, or only include the listed components.

[0089] In the present disclosure, the term "CD33" refers to CD33, a member of the sialoadhesin family, which is expressed on cells of the hematopoietic lineage, including myeloid precursors, monocytes, macrophages, dendritic cells, and mast cells, and on tumor cells associated with myeloproliferative or mast cell proliferative diseases, including acute myeloid leukemia and myelodysplastic syndrome, and leukemia stem cells.

[0090] The "antibody" described in the present disclosure is an immunoglobulin, and a complete antibody is usually a four-chain structure connected by inter-chain disulfide bonds, which is composed of two identical heavy chains and two identical light chains. The antigenicity of immunoglobulin heavy chain constant region is different due to the difference in amino acid composition and arrangement order. Accordingly, immunoglobulins can be divided into five types, or called isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and the corresponding heavy chains are μ chain, δ chain, γ chain, α chain and ε chain, respectively. The same class of Ig can be divided into different subclasses according to the difference in amino acid composition of hinge region and the number and position of heavy chain disulfide bonds, such as IgG can be divided into IgG1, IgG2, IgG3 and IgG4. The light chain is divided into κ chain or λ chain by the constant region. Each of the five types of Ig can have κ chain or λ chain.

[0091] The sequences of the heavy and light chains of an antibody, near the N-terminus, are highly variable, forming the variable region (Fv region); the remaining sequences are relatively stable, forming the constant region. The variable region includes three hypervariable regions (HVRs) and four relatively conserved framework regions (FRs). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity-determining region (CDR). Each light chain variable region (VL) and heavy chain variable region (VH) is composed of three CDR regions and four FR regions, arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus. The three CDR regions of the light chain are referred to as LCDR1, LCDR2 and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR3.

[0092] In the present 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 loops (CDRs) of that variable domain. In essence, it is the variable domain without the CDRs.

[0093] In the present specification, the term "complementarity-determining region", "CDR" or "hypervariable region" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contribute to antigen binding. Typically, there are three CDRs in each heavy chain variable region (HCDR1, HCDR2, HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, LCDR3). The boundaries of the amino acid sequences of the CDRs can be determined using any of a variety of well-known schemes, including the "Kabat" numbering convention (see Kabat et al. (1991) "Sequences of Proteins of Immunological Interest", 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering convention (see Al-Lazikani et al. (1997) JMB 273:927-948) and the ImMunoGenTics (IMGT) numbering convention (Lefranc M.P., Immunologist, 7, 132-136 (1999); Lefranc, M.P. et al., Dev. Comp. Immunol., 27, 55-77 (2003) and the like.

[0094] The antibodies of the present disclosure include murine, chimeric, humanized or fully human antibodies. In addition to full-length antibodies, the antibodies of the present disclosure also include antigen-binding fragments that are capable of binding to an antigen.

[0095] The term "murine antibody" in the present disclosure refers to a monoclonal antibody derived from a mouse prepared according to the knowledge and skill in the art. The preparation is performed by injecting a test subject with an antigen, and then isolating a hybridoma expressing an antibody having a sequence or functional property of interest. When the injected test subject is a mouse, the resulting antibody is a murine antibody.

[0096] The term "chimeric antibody" refers to an antibody in which the variable region of a murine antibody is fused with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody. To establish a chimeric antibody, a hybridoma secreting a murine-specific monoclonal antibody is first established, and then the variable region gene is cloned from the murine hybridoma cell. The constant region gene of a human antibody is cloned as needed, and the murine variable region gene is linked to the human constant region gene to form a chimeric gene, which is inserted into an expression vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic system or a prokaryotic system. The antibody light chain of the chimeric antibody further comprises a light chain constant region of a human κ, λ chain or a variant thereof. The antibody heavy chain of the chimeric antibody further comprises a heavy chain constant region of a human IgG1, IgG2, IgG3, IgG4 or a variant thereof, preferably a human IgG1, IgG2 or IgG4 heavy chain constant region, or a variant of the IgG1, IgG2 or IgG4 heavy chain constant region using amino acid mutations (such as YTE mutations or back mutations, L234A and / or L235A mutations, or S228P mutations).

[0097] The term "humanized antibody" also referred to as CDR-grafted antibody, refers to an antibody in which the CDR sequences of a murine antibody are grafted into a human antibody variable region framework, i.e., an antibody generated from different types of human germline antibody framework sequences. This can overcome the heterologous reaction induced by the chimeric antibody due to the presence of a large amount of murine protein components. Such framework sequences can be obtained from public DNA databases or published references including germline antibody gene sequences. For example, the germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet 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 the decrease in immunogenicity while causing a decrease in activity, the human antibody variable region framework sequence can be subjected to minimal back mutations or back mutations to maintain activity. The humanized antibody of the present disclosure also includes a humanized antibody further subjected to affinity maturation mutations of CDRs by yeast display.

[0098] 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, the constant region also is derived from human germline immunoglobulin sequences. A fully human antibody can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, a "fully human antibody" herein is not intended to include antibodies in which the CDR sequences are derived from another mammalian species (e.g., mouse).

[0099] Grafting of CDRs can result in a decrease in the affinity of the resulting antibody or antigen binding fragment thereof for the antigen due to framework residues that contact the antigen. Such interactions can be the result of somatic hypermutation. Thus, it can still be desirable to graft such donor framework amino acids into the framework of the humanized antibody. Amino acid residues from the non-human antibody or antigen binding fragment thereof that are involved in antigen binding can be identified by examining animal monoclonal antibody variable region sequences and structures. Each residue in the CDR donor framework that differs from germline can be considered relevant. If the closest germline cannot be determined, then the sequence can be compared to a subclass consensus sequence or to a consensus sequence of animal antibody sequences with a high percentage of similarity. Rare framework residues are considered likely to be the result of somatic hypermutation and thus play an important role in binding.

[0100] "Human antibody" (HuMAb), "humanized antibody", "fully human antibody", "fully human antibody" can be used interchangeably and can be an antibody derived from a human or an antibody obtained from a transgenic organism that has been "engineered" to produce specific human antibodies in response to antigenic challenge and can be produced by any method known in the art. In certain technologies, elements of the human heavy and light chain loci are introduced into a cell strain of an organism of the same species as the desired human antibody, such that the endogenous

[0101] The term "antigen-binding fragment" or "functional fragment" of an antibody refers to one or more fragments of an antibody that retain its ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VH and VL domains of a single arm of an antibody; (v) a dsFv, a stabilized antigen-binding fragment of a VHand VLformed by an interchain disulfide bond; (vi) diabodies, bispecific antibodies, and multispecific antibodies comprising a scFv, a dsFv, a Fab, and the like fragments. Moreover, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be produced as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., 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 of skill in the art, and are screened for utility in the same fashion as are intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulin. The antibody can be an antibody of different isotypes, e.g., an IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibody.

[0102] Fab is an antigen-binding fragment having a molecular weight of about 50,000 and having antigen-binding activity, which is obtained in a fragment by treating an IgG antibody molecule with a protease papain (which cleaves the amino acid residues of the H chain) in which about one-half of the H chain N-terminal side and the entire L chain are bound together by disulfide bonds.

[0103] F(ab')2 is an antigen-binding fragment having a molecular weight of about 100,000 and having antigen-binding activity and containing two Fab regions connected at the hinge position, which is obtained by digesting the lower portion of two disulfide bonds in the hinge region of IgG with the enzyme pepsin.

[0104] Fab' is an antigen binding fragment having a molecular weight of about 50,000 and antigen binding activity obtained by cleaving the disulfide bond of the hinge region of the above F(ab')2. The Fab' of the present disclosure can be produced by treating F(ab')2 that specifically recognizes and binds to an antigen with a reducing agent such as dithiothreitol.

[0105] In addition, the Fab' can be produced by inserting DNA encoding the Fab' fragment of the antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryote or a eukaryote to express the Fab'.

[0106] The term "single-chain antibody", "single-chain Fv" or "scFv" means a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) joined by a linker. Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, for example using 1-4 repeats of the variant (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present disclosure 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.

[0107] In the present disclosure, the term "specifically binds" or "selectively binds" means that the antibody binds to an epitope on a predetermined antigen.

[0108] In the present disclosure, the term "epitope" means a site on an antigen to which an immunoglobulin or antibody specifically binds. An epitope can be formed by contiguous amino acids, or non-contiguous amino acids juxtaposed by protein folding. Epitopes formed from contiguous amino acids are typically conserved when exposed to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon denaturing solvents. Epitopes typically include at least 3-15 amino acids in a unique spatial conformation. Methods of determining what epitope is bound by a given antibody are well known in the art, including immunoblot and immunoprecipitation detection assays, etc. Methods of determining the spatial conformation of an epitope include techniques in the art, such as X-ray crystallography and two-dimensional nuclear magnetic resonance, etc.

[0109] In the present disclosure, the terms "polypeptide", "protein", "peptide" are used interchangeably herein to refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having otherwise similar properties. A protein can be encoded by a gene.

[0110] In the present disclosure, the terms "polynucleotide", "nucleic acid" are used interchangeably to refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or their analogs. Polynucleotides can have any three-dimensional structure, and can perform any function for which polynucleotides can be employed, whether known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, an exon, an intron, 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. A nucleic acid molecule can be linear or circular.

[0111] In the present disclosure, the three letter code and one letter code of amino acids are used as described in J. Biol. Chem, 243, p3558 (1968).

[0112] In the present disclosure, "identity" refers to sequence similarity of two polynucleotide sequences or of two polypeptides. When a position in both of the compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent 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 in the shorter of the two sequences (i.e., fewer positions) times 100%. For example, if 6 of 10 positions in two sequences are matched or homologous, then the two sequences are 60% homologous. Generally, when comparing two sequences, the comparison is performed over a window of comparison, and the window is moved over the two sequences to determine the maximum percent identity.

[0113] In the present disclosure, the term "codon optimization" refers to a nucleotide sequence encoding a polypeptide has been configured to contain preferred codons for a host cell or organism to improve gene expression in the host cell or organism and increase translation efficiency.

[0114] In the present specification, "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector comprises cis-acting components sufficient for expression; other components for expression can be provided by the host cell or in an in vitro expression system. Expression vectors include all of those well known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses into which the recombinant polynucleotide is introduced (e.g., lentivirus, retrovirus, adenovirus, and adeno-associated virus). In the present disclosure, the term "CAR-T cell" refers to a chimeric antigen receptor T cell, which is usually composed of an extracellular targeting domain, a transmembrane region, and an intracellular signaling domain. The targeting domain of CAR is usually 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 to the target antigen; the transmembrane region is a hinge or spacer region that can anchor the single-chain variable domain to the cell membrane; the intracellular signaling domain is composed of a costimulatory factor and a CD3 signaling domain. When the antigen is recognized and bound, a stimulation signal is transmitted to the intracellular signaling domain, and the T cell is activated and exerts an effector function.

[0115] In the present disclosure, the term "chimeric antigen receptor (CAR)" is a fusion protein comprising an extracellular domain capable of binding to an antigen, a transmembrane domain derived from different polypeptides from the extracellular domain, and at least one intracellular domain. The specific molecules (such as antibodies) that recognize tumor cell surface antigens can be anchored on immune cells (such as T cells), allowing immune cells to recognize tumor antigens or viral antigens and kill tumor cells or viral cells. The "extracellular domain capable of binding to an antigen" refers to any oligopeptide or polypeptide capable of binding to an antigen. The "intracellular domain" refers to any oligopeptide or polypeptide known to function as a domain that transmits signals to activate or inhibit biological processes within cells.

[0116] In the present specification, the term "293F cell" is a cell line derived from 293 cells by genetic technology, and transient transfection of 293F cells is a convenient way to overexpress proteins and obtain intracellular and extracellular (secreted or membrane) proteins.

[0117] In the present specification, "administration," "administering," and "treatment" when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, 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. "Administration," "administering," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell includes contact of the reagent with the cell, as well as contact of the reagent with a fluid that is in contact with the cell. "Administration," "administering," and "treatment" also mean in vitro and ex vivo treatment of, for example, a cell by a reagent, diagnostic agent, binding composition, or by another cell. "Treatment" when applied to a human, veterinary, or research subject, refers to therapeutic, prophylactic, or preventative measures, research, and diagnostic applications.

[0118] In the present disclosure, the term "treatment" refers to a method of alleviating or eliminating a disorder and / or accompanying symptoms.

[0119] In the present disclosure, the term "tumor" refers to a neoplasm or solid lesion formed by abnormal cell growth, optionally, the tumor cell is Molm13-Luciferase.

[0120] In the present disclosure, the term "transfection" refers to the entry of a recombinant plasmid vector or a free nucleotide into a eukaryotic cell mediated by liposomes or the like.

[0121] In the present disclosure, the term "infection" refers to a process in which a recombinant viral vector invades a recipient cell in a gene transfer experiment. A CAR-T cell binds to a specific antigen through a CAR, thereby transmitting a signal into the cell, and the cell is thus activated. The activation of a cell expressing a CAR varies depending on the kind of the host cell and the intracellular domain of the CAR, and can be confirmed based on, for example, the release of a cytokine, the improvement of the cell proliferation rate, the change of a cell surface molecule, etc., as an index. For example, the release of a cytokine (tumor necrosis factor, lymphotoxin, etc.) from an activated cell causes the destruction of a cell expressing a targeted antigen. In addition, the release of a cytokine or the change of a cell surface molecule stimulates other immune cells, such as B cells, dendritic cells, NK cells, and macrophages.

[0122] In the present specification, the term "tag" refers to a short peptide that is fused or linked to a protein of interest (e.g., an antibody of the present disclosure) and thereby facilitates the soluble expression, detection, and / or purification of the recombinant protein. The tag can be fused or linked to the N- and / or C-terminus of the protein of interest (optionally through a linker or a protease cleavage site).

[0123] In the present specification, the terms "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny. The term "transformant" and "transformed cell" includes the primary subject cell and cultures derived from it, regardless of the number of transfers. It is also understood that all progeny can not necessarily be identical to the parent cell since recombination or mutation can occur during proliferation and propagation.

[0124] In the present disclosure, "multispecific antibodies" include antibodies or antigen-binding fragments thereof targeting CD33 as described above. The multispecific antibodies further include antigen-binding fragments targeting other antigens.

[0125] In the present specification, "diagnosing" includes detection or identification of a disease state or condition in a subject, determining the likelihood that a subject will develop a given disease or condition, determining the likelihood that a subject with a disease or condition will respond to treatment, determining the prognosis (or likely 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.

[0126] In the present specification, "treatment" means administering to a patient, either internally or externally, a therapeutic agent, such as an antibody of the present disclosure, having one or more symptoms of a disease, which is known to have a therapeutic effect on these symptoms. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the treated patient or population, whether by inducing regression of such symptoms or inhibiting development of such symptoms to any clinically measurable extent. The amount of therapeutic agent effective for alleviating any particular symptom of a disease (also referred to as "therapeutically effective amount") can vary according to factors such as the disease state, age, and weight of the patient, and the ability of the drug to elicit a desired effect in the patient. Whether a disease symptom has been alleviated can be assessed by any clinical detection method typically used by a physician or other professional health care provider to assess the severity or progression of the symptom.

[0127] In the present specification, "pharmaceutical composition" means containing one or more antibodies described herein, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, to facilitate absorption of the active ingredient, and to thereby elicit a biological activity.

[0128] In the present specification, the term "pharmaceutically acceptable" (or "pharmacologically acceptable") refers to molecular entities and compositions that, where appropriate, are nontoxic to animals or humans at the dosages and concentrations employed, do not elicit adverse reactions, allergic reactions or other untoward effects. The term "pharmaceutically acceptable carrier" as used herein encompasses any and all solvents, dispersion media, coatings, antibacterial agents, isotonic and absorption delaying agents, buffers, excipients, binders, lubricants, gels, surfactants, and the like that can be used with a pharmaceutically acceptable substance as a medium.

[0129] The technical solutions of the present disclosure are described in detail as follows:

[0130] Antibodies or antigen-binding fragments thereof targeting CD33

[0131] According to some embodiments of the present disclosure, there are provided antibodies or antigen-binding fragments thereof targeting CD33, comprising a heavy chain variable region or a light chain variable region, each of which comprises three complementarity determining regions (CDRs): LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3.

[0132] In some embodiments, the LCDR1 comprises an amino acid sequence as set forth 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 more sequence identity to any one of SEQ ID NO: 6 or SEQ ID NO: 13 or SEQ ID NO: 20 or SEQ ID NO: 27.

[0133] In some embodiments, the LCDR2 comprises an amino acid sequence as set forth in YAS or YTS, or a variant thereof having at least 70% or 99% or more sequence identity to any one of the YAS and YTS amino acid sequences.

[0134] In some embodiments, the LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 7, SEQ ID NO: 14, SEQ ID NO: 21, SEQ ID NO: 28, SEQ ID NO: 32, or a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% or more sequence identity to any one of SEQ ID NO: 7, SEQ ID NO: 14, SEQ ID NO: 21, SEQ ID NO: 28, SEQ ID NO: 32.

[0135] In some embodiments, the HCDR1 comprises an amino acid sequence as set forth 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 more sequence identity to any one of SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 16, or SEQ ID NO: 23.

[0136] In some embodiments, the LCDR2 comprises an amino acid sequence 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, or a variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more sequence identity to any one of SEQ ID NO: 3, SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 24, and SEQ ID NO: 30.

[0137] In some embodiments, the LCDR3 comprises an amino acid sequence as set forth 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 more sequence identity to any one of SEQ ID NO: 4 SEQ ID NO: 11, SEQ ID NO: 18, and SEQ ID NO: 25.

[0138] Further, the antibody or antigen binding fragment thereof targeting CD33, the heavy chain variable region thereof comprises:

[0139] (a1) an LCDR1 as set forth in SEQ ID NO: 6, an LCDR2 as set forth in SEQ ID NO: 7, an LCDR3 as set forth in SEQ ID NO: 8;

[0140] (a2) an LCDR1 as set forth in SEQ ID NO: 13, an LCDR2 as set forth in the sequence YTS, an LCDR3 as set forth in SEQ ID NO: 14;

[0141] (a3) an LCDR1 as set forth in SEQ ID NO: 20, an LCDR2 as set forth in the sequence YTS, an LCDR3 as set forth in SEQ ID NO: 21;

[0142] (a4) LCDR1 as set forth in SEQ ID NO: 27, LCDR2 as the sequence YTS, LCDR3 as set forth in SEQ ID NO: 28;

[0143] (a5) LCDR1 as set forth in SEQ ID NO: 13, LCDR2 as the sequence YTS, LCDR3 as set forth in SEQ ID NO: 32;

[0144] the heavy chain variable region thereof comprises:

[0145] (b1) HCDR1 as set forth in SEQ ID NO: 2, HCDR2 as set forth in SEQ ID NO: 3, HCDR3 as set forth in SEQ ID NO: 4;

[0146] (b2) HCDR1 as set forth in SEQ ID NO: 9, HCDR2 as set forth in SEQ ID NO: 10, HCDR3 as set forth in SEQ ID NO: 11;

[0147] (b3) HCDR1 as set forth in SEQ ID NO: 16, HCDR2 as set forth in SEQ ID NO: 17, HCDR3 as set forth in SEQ ID NO: 18;

[0148] (b4) HCDR1 as set forth in SEQ ID NO: 23, HCDR2 as set forth in SEQ ID NO: 24, HCDR3 as set forth in SEQ ID NO: 25;

[0149] (b5) HCDR1 as set forth in SEQ ID NO: 9, HCDR2 as set forth in SEQ ID NO: 30, HCDR3 as set forth in SEQ ID NO: 11.

[0150] In some alternative embodiments, 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.

[0151] In some exemplary embodiments, the LCDR1, LCDR2, LCDR3, or HCDR1, HCDR2, and HCDR3 are separated by framework regions FR1, FR2, FR3, and FR4.

[0152] In some alternative embodiments, the framework regions FR are human, murine, rabbit, or camel.

[0153] In some preferred embodiments, the framework regions FR are murine FR regions.

[0154] In some alternative embodiments, the antibody or antigen-binding fragment thereof targeting CD33 comprises a murine antibody or fragment thereof, wherein the light chain variable region of the murine antibody or fragment thereof comprises one or more of the following (i)-(iii):

[0155] (i) an amino acid sequence as set forth in any one of SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 19, SEQ ID NO: 26, SEQ ID NO: 31;

[0156] (ii) a sequence having at least 97%, 98%, or 99% identity to any one of the amino acid sequences as set forth in SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 19, SEQ ID NO: 26, SEQ ID NO: 31;

[0157] (iii) an amino acid sequence adding, substituting, deleting, or inserting 1 or more amino acid residues in the amino acid sequence as set forth in SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 19, SEQ ID NO: 26, SEQ ID NO: 31, and which retains the function of the amino acid sequence as set forth in SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 19, SEQ ID NO: 26, SEQ ID NO: 31.

[0158] wherein the heavy chain variable region of the murine antibody or fragment thereof comprises one or more of the following (iv)-(vi):

[0159] (iv) a sequence as set forth in any one of SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 29;

[0160] (v) a sequence having at least 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 29;

[0161] (vi) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted, or inserted in the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 29, and which retains the function of the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 29.

[0162] In other preferred embodiments, the framework region FR comprises a human-derived FR region. Optionally, the CD33-targeting antibody or antigen-binding fragment thereof is partially or fully humanized.

[0163] In some optional embodiments, the CD33-targeting antibody or antigen-binding fragment thereof comprises a humanized antibody or fragment thereof, wherein the light chain variable region of the humanized antibody or fragment thereof comprises one or more of the following (vii)-(ix):

[0164] (vii) the amino acid sequence set forth in SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 42;

[0165] (viii) a sequence having at least 97%, 98%, or 99% identity to any one of the amino acid sequences set forth in SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 42;

[0166] (ix) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted, or inserted in the amino acid sequence set forth in SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 42, and which retains the function of the amino acid sequence set forth in SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 42.

[0167] wherein the heavy chain variable region of the humanized antibody or fragment thereof comprises one or more of the following (x)-(xii):

[0168] (x) the sequence set forth in any one of SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NO: 41;

[0169] (xi) a sequence having at least 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NO: 41;

[0170] (xii) an amino acid sequence that adds, substitutes, deletes, or inserts one or more amino acid residues in the amino acid sequence set forth in SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NO: 41, and that retains the function of the amino acid sequence set forth in SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NO: 41.

[0171] In some embodiments, the antibody or antigen-binding fragment thereof targeting CD33 further comprises a heavy chain constant region derived from human IgGl, human IgG2, human IgG3, human IgG4, or a variant thereof; and / or, the antibody or antigen-binding fragment thereof targeting CD33 further comprises a light chain constant region derived from human kappa chain, lambda chain, or a variant thereof.

[0172] In some embodiments, the antigen-binding fragment is selected from at least one of F(ab)2, Fab', Fab, Fv, scFv, multispecific antibody, single-chain antibody.

[0173] The CD33-targeting antibody or antigen-binding fragment thereof of the present disclosure refers to a polypeptide having CD33-binding activity, including the above-mentioned CDR regions. The term also includes variant forms of the polypeptide containing the above-mentioned CDR regions having the same function as the CD33-targeting antibody or antigen-binding fragment thereof of the present disclosure. These variant forms include, but are not limited to, deletion, insertion and / or substitution of one or more (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids, and addition of one or a few (typically within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, substitution with similar or similar performance amino acids generally does not change the function of the protein. For another example, addition of one or a few amino acids at the C-terminus and / or N-terminus also generally does not change the function of the protein. Variant forms of single-domain antibodies include homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize to the coding DNA of the antibody of the present disclosure under high or low stringency conditions, and polypeptides or proteins obtained using antisera against the CD33-targeting antibody or antigen-binding fragment thereof of the present disclosure.

[0174] <Chimeric antigen receptor>

[0175] The present disclosure provides a chimeric antigen receptor comprising:

[0176] (A) an extracellular domain that specifically binds CD33;

[0177] (B) a transmembrane domain;

[0178] (C) an intracellular signaling domain.

[0179] (Extracellular domain)

[0180] In the present disclosure, the extracellular domain is an extracellular antigen binding domain composed of an antigen binding domain (ABD, scFv) and a hinge region (Hinge) that functions as a linker.

[0181] In some embodiments, the scFv is a single-chain variable fragment of a monoclonal antibody that recognizes and binds to an antigen.

[0182] In some embodiments, the antigen binding domain comprises the above-mentioned antibody or antigen-binding fragment thereof targeting CD33. Illustratively, the antigen binding domain comprises the above-mentioned humanized antibody or fragment thereof.

[0183] In the present disclosure, a "hinge region" is a domain that makes up IgG, IgA, and IgD class immunoglobulin molecules, located between CH1 and CH2, connecting the Fab and Fc segments, or the segment between the extracellular functional domain and the transmembrane region of membrane proteins such as CD8, CD4, PD1, etc. This segment is rich in proline, does not form an alpha helix, is prone to stretching and a certain degree of twisting, and is conducive to the complementary binding between the antigen binding site of the antibody and the epitope of the antigen.

[0184] (transmembrane domain)

[0185] In the present disclosure, the term "transmembrane domain" (also referred to as transmembrane region) can include one or more contiguous fragments of amino acids of the transmembrane region, for example, one or more amino acids associated with the extracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of 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 can be from the same protein from which the signaling domain, costimulatory domain, or hinge domain is derived. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domain to the transmembrane domain of the same or different surface membrane protein, for example, to minimize the interaction with other members of the receptor complex containing the transmembrane domain. In one aspect, the transmembrane domain is capable of homodimerization with another chimeric receptor on the surface of a cell expressing the chimeric receptor. In one aspect, the amino acid sequence of the transmembrane domain can be modified or substituted in order to minimize the interaction with the binding domain of the natural binding partner present in a cell expressing the same chimeric receptor. The transmembrane domain can be derived from a natural or recombinant source. When the source is natural, the domain can be derived from any membrane-bound protein or transmembrane protein. In one aspect, the transmembrane domain is capable of transmitting a signal to the intracellular domain as long as the chimeric receptor binds to the target antigen. The transmembrane domain used particularly in the present disclosure can include at least the following transmembrane domains: for example, the alpha, beta or zeta chain of the T-cell receptor, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 transmembrane domain.

[0186] (intracellular signaling domain)

[0187] In some embodiments, the intracellular signaling domain can comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from a molecule responsible for primary stimulation or antigen-dependent stimulation. In one embodiment, the intracellular signaling domain can comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from a molecule responsible for costimulatory signals or antigen-independent stimulation. For example, in the case of a CAR-T, the primary intracellular signaling domain can comprise the cytoplasmic sequence of a T cell receptor, and the costimulatory intracellular signaling domain can comprise the cytoplasmic sequence from a co-receptor or costimulatory molecule.

[0188] In some embodiments, the primary intracellular signaling domain can comprise a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of primary cytoplasmic signaling sequences comprising ITAMs include, but are not limited to, those derived from CD3-zeta, FcRy, FcRP, CD3y, CD35, CD3s, CD5, CD22, CD79a, CD79b, and CD66d DAP10, and DAP12.

[0189] In the present disclosure, “zeta” or alternatively “zeta chain,” “CD3-zeta,” “TCR-zeta,” or “CD3 zeta” is defined as the protein provided at GenBank No. BAG36664.1, or equivalent residues from a non-human species (e.g., murine, rabbit, primate, mouse, rodent, monkey, ape, etc.), and “zeta stimulatory domain” or alternatively “CD3-zeta stimulatory domain” or “TCR-zeta stimulatory domain” is defined as the amino acid residues of the cytoplasmic domain from the zeta chain sufficient to transmit the initial signal necessary to functionally deliver T cell activation.

[0190] In the present disclosure, “costimulatory molecule” refers to a cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response of the T cell, such as but not limited to proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for efficient immune responses. Costimulatory 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).

[0191] The costimulatory intracellular signaling domain can be an intracellular portion of a costimulatory molecule. The costimulatory molecule can be represented in the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), and activating 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 a ligand that specifically binds with CD83, among others.

[0192] In some embodiments of the present disclosure, 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.

[0193] <Isolated polynucleotide>

[0194] The present disclosure provides an isolated polynucleotide encoding the antibody or antigen binding fragment targeting CD33 described above.

[0195] In some embodiments, 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 DNA. The DNA can be a coding strand or a non-coding strand. Unless otherwise specified, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.

[0196] <Expression cassette, recombinant vector, host cell>

[0197] Based on the polynucleotide obtained according to the present disclosure, an expression cassette comprising the polynucleotide as described in the present disclosure is provided.

[0198] In other embodiments, the present disclosure provides a recombinant vector comprising the polynucleotide or expression cassette as described above.

[0199] In other embodiments, the present disclosure provides a host cell comprising the polynucleotide, expression cassette, or recombinant vector as described in the present disclosure.

[0200] In some specific embodiments, the host cell is a bacterium; illustratively, the host cell is Escherichia coli, Bacillus subtilis, but not limited thereto.

[0201] In some specific embodiments, the host cell is a bacterium; illustratively, the host cell is Escherichia coli, Bacillus subtilis, but not limited thereto.

[0202] In some specific embodiments, the host cell is a bacterium; illustratively, the host cell is Escherichia coli, Bacillus subtilis, but not limited thereto.

[0203] <Recombinant immune cell>

[0204] The present disclosure provides a recombinant immune cell comprising the chimeric antigen receptor described above.

[0205] The recombinant immune cell described in the present disclosure can bind to a CD33-expressing cell and have a specific killing effect on the CD33-expressing cell.

[0206] In some specific embodiments, the CD33-expressing cell includes a Molm13 cell line expressing CD33.

[0207] In some embodiments, the recombinant immune cell is an immune cell derived from a mammal.

[0208] The mammal includes, but is not limited to, primates (e.g., humans, monkeys), cows, sheep, goats, alpacas, horses, dogs, cats, rabbits, rats, mice, etc.

[0209] In the present disclosure, there is no particular limitation in principle to the kind of immune cell. In some embodiments, the immune cell is selected from one or more of T cells, B cells, NK cells, mast cells, tumor infiltrating lymphocytes. In some preferred embodiments, the immune cell is selected from T cells or NK cells. In some specific embodiments, the T cell is 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 a double negative T cell or a NKT cell. In some more preferred embodiments, the T cell is a CD8 + T cell.

[0210] In some specific embodiments, the recombinant immune cell is a recombinant T cell.

[0211] <COMPOSITION>

[0212] The present disclosure also provides a composition comprising at least one of the CD33-targeting antibody or antigen-binding fragment thereof, polynucleotide, vector, recombinant cell, chimeric antigen receptor, or recombinant immune cell described above.

[0213] In some alternative embodiments, the composition comprises a pharmaceutical composition.

[0214] Further, the pharmaceutical composition can further comprise a pharmaceutically acceptable carrier and / or adjuvant. In some embodiments, the pharmaceutically acceptable carrier and / or adjuvant 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 in the case of oral administration, and the pharmaceutical composition thus formulated can be administered to the subject by any appropriate means known to those skilled in the art as needed.

[0215] In some embodiments, the pharmaceutical composition can be prepared in any one of the pharmaceutically conventional dosage forms.

[0216] <AGENT, KIT, METHOD FOR DETECTION, DIAGNOSIS, PREVENTION, OR TREATMENT>

[0217] The present disclosure provides an agent or a kit comprising at least one of the CD33-targeting antibody or antigen-binding fragment thereof, polynucleotide, vector, recombinant cell, chimeric antigen receptor, or recombinant immune cell, and composition described above. The agent or kit can be used for the detection and / or diagnosis of CD33. The agent or kit can also be used for the prevention or treatment of a CD33-positive tumor, which includes having an elevated level of CD33 at the nucleic acid or protein level.

[0218] In another embodiment of the present disclosure, a method for detecting a CD33 protein in a sample is provided, which comprises:

[0219] (a) contacting the sample with the CD33-targeting antibody or antigen-binding fragment thereof as described above;

[0220] (b) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of CD33 protein in the sample.

[0221] In some alternative embodiments, the method can be a method for detecting CD33 protein in vitro in a non-diagnostic assay, which corresponds to performing step (a) above in vitro.

[0222] In some alternative embodiments, the method can be a method for detecting CD33 protein in vitro in a non-diagnostic assay, which corresponds to performing step (a) above in vitro.

[0223] In some alternative embodiments, the method can be a method for detecting CD33 protein in vitro in a non-diagnostic assay, which corresponds to performing step (a) above in vitro.

[0224] In some preferred embodiments, the tumor is a cancer is a myeloid leukemia.

[0225] <Multi-specific antibody>

[0226] The present disclosure provides a multi-specific antibody comprising the CD33-targeting antibody or antigen-binding fragment thereof as described above. In some embodiments, the multi-specific antibody further comprises an antigen-binding fragment targeting other antigens, for example, comprising a domain of anti-CD3, which can simultaneously target CD33 of a cell surface antigen and CD3 of a T cell surface triggering molecule, so as to activate the T cell, resulting in the effect of killing the target cell. The domain of anti-CD3 can be a single-chain antibody or an antibody Fab fragment, etc.

[0227] <Antibody conjugate>

[0228] In some embodiments, the present disclosure provides an antibody conjugate comprising the CD33-targeting antibody or antigen-binding fragment thereof as described above. In some embodiments, the CD33-targeting antibody or antigen-binding fragment thereof can be conjugated with an enzyme label, a fluorescent label, a biotin label to obtain an antibody conjugate. In some exemplary embodiments, the antibody conjugate further comprises a cytotoxic drug.

[0229] In the present disclosure, the term "cytotoxic drug" generally refers to a toxic drug, which can be a chemical molecule having a strong destructive effect on the normal growth of tumor cells. The cytotoxic drug can kill tumor cells at a sufficiently high concentration.

[0230] The "cytotoxic drug" can include a toxin, such as a small molecule toxin or an enzymatically active toxin of bacterial, fungal, plant or animal origin, a radioisotope (for example, At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, or a radioisotope of Lu), a toxic drug, a chemotherapeutic agent, an antibiotic or a nucleolytic enzyme, or a derivative thereof.

[0231] Alternatively, the cytotoxic drug is a microtubulin inhibitor and a DNA topoisomerase inhibitor or other cytotoxic compound.

[0232] In some embodiments, the cytotoxic drug is MMAE.

[0233] Examples

[0234] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description, but it is to be understood that both the detailed description and the specific examples, while indicating specific embodiments of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present disclosure, will become apparent to those skilled in the art from this detailed description.

[0235] The experimental techniques and experimental methods used in the present examples are all conventional techniques and methods, and are not specifically described unless otherwise specified. For example, the experimental methods not specifically described in the following examples are generally performed according to the conditions 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. The materials, reagents, etc. used in the examples can be obtained through regular commercial channels unless otherwise specified.

[0236] Example 1. Screening of murine monoclonal antibodies

[0237] 1. Preparation of human CD33 antigen

[0238] The extracellular segment sequence information of human CD33 was retrieved from UniProt database (UniProt Accession#P20138), and the sequence from the 18th Asp to the 259th His was selected. A His tag was added at the C-terminal of the sequence, and the codons were optimized according to the codon preference of human. After gene synthesis, the gene was subcloned into the eukaryotic expression vector pcDNA3.4. After Sanger sequencing verification, the plasmid was extracted for standby use. The constructed protein eukaryotic expression vector was transiently transfected into 293F cells, and the protein expression supernatant was collected and subjected to nickel column purification of the target protein. SDS-PAGE experiment was performed to detect the purity of the protein, and Nanodrop 2000 was used to determine the protein concentration. The purified protein was used for immunization and antibody screening.

[0239] 2. Construction of CHO-K1-CD33 cell strain

[0240] According to 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 lentivirus expression vector to construct the Lenti-CMV-CD33-puro overexpression vector. After packaging the lentivirus, 100 μL of the lentivirus was added to CHO-K1 cells, and 48 h later, 8 μg / ml puromycin was added for the screening of CHO-K1-CD33 cell strain. After the completion of cell strain screening, the expression of human CD33 on the cell membrane was confirmed by FACS using positive antibodies.

[0241] 3. Mouse immunization

[0242] All mice were raised in a barrier system and fed with sterilized pellet feed and autoclaved drinking water. Five Balb / c mice (SPF level) were labeled with ear tags, and 100 μg of human CD33 protein was used for each mouse. After emulsification with Freund's adjuvant, the mice were immunized subcutaneously in multiple points, a total of 3 times (the first immunization used complete Freund's adjuvant; all subsequent immunizations used incomplete Freund's adjuvant), with an interval of 14 days. After the completion of immunization, peripheral blood was collected from the tail vein, and serum was separated for FACS detection of the immune titer.

[0243] 4. Hybridoma fusion and monoclonal screening

[0244] 1) The mouse with good immune titer was subjected to impact immunization for 3 days, and the mouse spleen was obtained under sterile conditions to prepare a B cell single cell suspension. The B cell single cell suspension was mixed with SP2 / 0 myeloma cells at a ratio of 1:1, and cell fusion was performed using a BTX cell electrofusion instrument.

[0245] 2) After electrofusion, all cells were immediately suspended in complete culture medium (DMEM, 20% FBS and HAT), and inoculated into 96-well plates.

[0246] 3) About 10 days after fusion, change to HT medium. After two days of culture, take the supernatant for ELISA to detect the binding with the target antigen human CD33, and use FACS to detect the binding with CHO-K1-CD33 cells. After each round of subcloning, perform ELISA and FACS experiments according to the same procedure until a single clone is formed.

[0247] 5. Hybridoma sequencing

[0248] Take 1 x 10 6 cells obtained from the final screening, use Trizol to lyse, extract total RNA from the hybridoma cells according to the standard method, reverse transcribe into cDNA, use hybridoma sequencing primers to amplify the heavy and light chain variable regions of the antibody by PCR, then perform TA cloning, subclone the PCR obtained fragments into T vector, pick clones for sequencing. Analyze the final obtained antibody sequence.

[0249] 6. Hybridoma sequencing results

[0250] Sequence the positive hybridoma monoclonal cells, and obtain the heavy and light chain variable region sequence information of the positive clones as follows, where the underlined part is the CDR region of the antibody.

[0251] >Clone 2VH(SEQ ID NO:1)

[0252] CDR1: GYAFTNYL (SEQ ID NO: 2)

[0253] CDR2: INPGSGDT (SEQ ID NO: 3)

[0254] CDR3: ARSPNYYGSSYFDY (SEQ ID NO: 4)

[0255] >Clone 2VL(SEQ ID NO:5)

[0256] CDR1: QNVNYY (SEQ ID NO: 6)

[0257] CDR2: YAS

[0258] CDR3: QQDYNSPYT (SEQ ID NO: 7)

[0259] >Clone 3 VH(SEQ ID NO:8)

[0260] CDR1 : GYTFTRYW (SEQ ID NO: 9)

[0261] CDR2: INPSNGQT (SEQ ID NO: 10)

[0262] CDR3: ARWHYGLDY (SEQ ID NO: 11)

[0263] > Clone 3 VL (SEQ ID NO: 12)

[0264] CDR1 : QDINKY (SEQ ID NO: 13)

[0265] CDR2: YTS

[0266] CDR3: LHYDNLLT (SEQ ID NO: 14)

[0267] > Clone 5 VH (SEQ ID NO: 15)

[0268] CDR1 : GFSLNTSGLG (SEQ ID NO: 16) CDR2: IWWDDIK (SEQ ID NO: 17)

[0269] CDR3: ARRGHNNAMDY (SEQ ID NO: 18)

[0270] > Clone 5 VL (SEQ ID NO: 19)

[0271] CDR1 : QDINQY (SEQ ID NO: 20)

[0272] CDR2: YTS

[0273] CDR3: LHYGNLLWT (SEQ ID NO: 21)

[0274] > Clone 6 VH (SEQ ID NO: 22)

[0275] CDR1 : GFTFSNYA (SEQ ID NO: 23)

[0276] CDR2: ISSGGDT (SEQ ID NO: 24)

[0277] CDR3: VRGEANWDYFDY (SEQ ID NO: 25)

[0278] Clone 6 VL (SEQ ID NO: 26)

[0279] CDR1 : QDISNY (SEQ ID NO: 27)

[0280] CDR2: YTS

[0281] CDR3: QQGDTLPWT (SEQ ID NO: 28)

[0282] Clone 7 VH (SEQ ID NO: 29)

[0283] CDR2: IKPSDGRT (SEQ ID NO: 30)

[0284] CDR3: ARWHYGLDY (SEQ ID NO: 11)

[0285] Clone 7 VL (SEQ ID NO: 31)

[0286] CDR1 : QDINKY (SEQ ID NO: 13)

[0287] CDR2: YTS

[0288] CDR3: LHYENLLT (SEQ ID NO: 32)

[0289] Example 2. Antibody humanization design and affinity testing

[0290] According to the obtained amino acid sequence information of the mouse anti-heavy chain and light chain, the CDR region sequence of the original antibody is retained unchanged, and according to the results of germline alignment and antibody structure simulation, different human antibody templates are selected for the heavy chain and the light chain respectively, and back mutation is performed in the framework region after humanization to design candidate humanized antibody sequences. The heavy chain and light chain of the humanized antibody are respectively synthesized by gene synthesis, the heavy chain is subcloned into the pcDNA3.4-hIgG1 expression vector to construct an antibody heavy chain expression vector, and the light chain is subcloned into the pcDNA3.4-hIgKc expression vector to construct an antibody light chain expression vector. After the vector is sequenced and verified to be correct, 293F cells are transiently transfected, the culture medium supernatant is collected, and the recombinant humanized antibody is purified by Protein A, and FACS is used to monitor the binding of Clone2-HM, Clone3-HM, Clone6-HM, Clone7-HM and recombinant CHO-CD33 cells overexpressing the target gene CD33. The hIgG1 heavy chain constant region amino acid sequence (SEQ ID NO: 43):

[0291] The hIgKc light chain constant region amino acid sequence (SEQ ID NO: 44):

[0292] The heavy chain and light chain variable region sequence information of the humanized antibody sequence is as follows, wherein the part indicated by the underline is the CDR region of the antibody.

[0293] >Clone2_VH-HM (SEQ ID NO: 33)

[0294] CDR1: GYAFTNYL (SEQ ID NO: 2)

[0295] CDR2: INPGSGDT (SEQ ID NO: 3)

[0296] CDR3: ARSPNYYGSSYFDY (SEQ ID NO: 4)

[0297] >Clone2_VL-HM (SEQ ID NO: 34)

[0298] CDR1: QNVNYY (SEQ ID NO: 6)

[0299] CDR2: YAS

[0300] CDR3: QQDYNSPYT (SEQ ID NO: 7)

[0301] Clone3_VH-HM (SEQ ID NO: 35)

[0302] CDR1 : GYTFTRYW (SEQ ID NO: 9)

[0303] CDR2: INPSNGQT (SEQ ID NO: 10)

[0304] CDR3: ARWHYGLDY (SEQ ID NO: 11)

[0305] Clone3_VL-HM (SEQ ID NO: 36)

[0306] CDR1 : QDINKY (SEQ ID NO: 13)

[0307] CDR2: YTS

[0308] CDR3: LHYDNLLT (SEQ ID NO: 14)

[0309] Clone5_VH-HM (SEQ ID NO: 37)

[0310] CDR1 : GFSLNTSGLG (SEQ ID NO: 16)

[0311] CDR2: IWWDDIK (SEQ ID NO: 17)

[0312] CDR3: ARRGHNNAMDY (SEQ ID NO: 18)

[0313] Clone5_VL-HM (SEQ ID NO: 38)

[0314] CDR1 : QDINQY (SEQ ID NO: 20)

[0315] CDR2: YTS

[0316] CDR3: LHYGNLLWT (SEQ ID NO: 21)

[0317] Clone6_VL-HM (SEQ ID NO: 39)

[0318] CDR1 : QDISNY (SEQ ID NO: 27)

[0319] CDR2: YTS

[0320] CDR3: QQGDTLPWT (SEQ ID NO: 28)

[0321] >Clone6_VH-HM (SEQ ID NO: 40)

[0322] CDR1: GFTFSNYA (SEQ ID NO: 23)

[0323] CDR2: ISSGGDT (SEQ ID NO: 24)

[0324] CDR3: VRGEANWDYFDY (SEQ ID NO: 25)

[0325] >Clone7_VH-HM (SEQ ID NO: 41)

[0326] CDR1: GYTFTRYW (SEQ ID NO: 9)

[0327] CDR2: IKPSDGRT (SEQ ID NO: 30)

[0328] CDR3: ARWHYGLDY (SEQ ID NO: 11)

[0329] >Clone7_VL-HM (SEQ ID NO: 42)

[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 detection, and the results are shown in Table 1.

[0334] Table 1

[0335] Example 3. Killing of target cells by T cells expressing candidate antibody chimeric antigen receptors

[0336] 1. Preparation of CAR-T cells

[0337] Clone2HM, Clone3HM, Clone5HM, Clone6HM, Clone7HM were selected according to the humanization detection results, and chimeric antigen receptor lentiviral expression vectors (activation signals were 4-1BB and CD3Zeta) were constructed, CAR lentiviral expression vectors were constructed, after the preparation of recombinant lentivirus, primary T cells were infected, and CAR-T cells were prepared for pharmacodynamic evaluation.

[0338] >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 bold part is the 4-1BB intracellular signal domain sequence, the italic part is the CD3Zeta intracellular signal domain, and the remaining part is the antibody light chain variable region and heavy chain variable region sequence)

[0339] >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 bold part is the 4-1BB intracellular signal domain sequence, the italic 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] >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 bold part is the 4-1BB intracellular signal domain sequence, the italic part is the CD3Zeta intracellular signal domain, and the remaining part is the antibody light chain variable region and heavy chain variable region sequence)

[0341] >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 bold part is the 4-1BB intracellular signal domain sequence, the italic 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] >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 bold part is the 4-1BB intracellular signal domain sequence, the italic part is the CD3Zeta intracellular signal domain, and the remaining part is the antibody light chain variable region and heavy chain variable region sequence)

[0343] 2. Lysis of target cells by CAR-T cells

[0344] 1). Adjust the Molm13-Luciferase target cell state to the logarithmic growth phase, resuspend the target cells in complete culture medium, and adjust the cell density to 2 x 10 5 6 x 105 / mL. Take a new 96-well plate, and inoculate the target cells in accordance with 100% of the amount of the wells. Add 100 μL of sterile water to the unused wells in the 96-well plate every four weeks to prevent water evaporation in the experimental wells. Place the plate in a 5% CO2, 37C incubator and incubate overnight.

[0345] 2). Centrifuge to collect the prepared CAR-T cells, resuspend using 10% FBS in 1640 culture medium; remove the 96-well plate from the incubator, completely aspirate the culture medium in the wells, gently wash the cells with sterile PBS once, then add CAR-T cells according to different CAR-T cell and target cell ratios, and supplement the final volume to 200 μL / well; Maxi lysis is to inoculate the same number of target cells, but without CAR-T cells. Place the plate in a 5% CO2, 37C incubator and incubate for 18 hours.

[0346] 3). After the incubation is complete, remove the plate from the incubator, and reflect the lysis ability of the recombinant CAR-T cells on the target cells by detecting the Luciferase activity. At the same time, collect the culture supernatant after co-culture, and use ELISA to detect the change in the expression level of the cytokine secretion after the CAR-T cells are activated.

[0347] 4). Target cell lysis percentage calculation formula:

[0348] RLU 样本 represents the fluorescence intensity of the sample co-cultured with different CAR-T cells and target cells, RLU 最大值 represents the fluorescence intensity of the sample cultured only with target cells.

[0349] Results:

[0350] Using Molm13-Luciferase as the target cells and CAR-T cells expressing different humanized antibodies as the effector cells, the in vitro co-culture system was established according to the effector-target ratio of FIG. 3. The results showed that the CAR-T cells constructed using the candidate antibodies in the present disclosure had strong killing effect on Molm13-Luciferase cells compared with the control T; and the killing effect was consistent with that of the CAR-T cells based on the positive control antibody Gentuzumab.

[0351] Using Molm13-Luc as the target cell and the prepared CAR-T cells as the effector cells, in vitro co-culture systems were established according to the effector-target ratios of 1:1, 2.5:1, 5:1, and 10:1 as shown in Figure 4. The results showed that, compared with blank T cells, CAR-T cells stimulated by Molm13-Luciferase cells had significantly enhanced IFN-γ secretion, which was consistent with CAR-T cells constructed based on the positive control antibody Gemtuzumab.

[0352] Example 4. CAR in vivo efficacy verification

[0353] 1. Animal modeling and grouping

[0354] 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.

[0355] 2. Experimental Observation

[0356] 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.

[0357] According to the imaging results shown in Figure 5, the CAR-T cells constructed based on the humanized antibody disclosed in this paper can significantly inhibit the growth of tumor cell lines in mice.

[0358] Example 5. Killing effect of candidate antibody on target cells after conjugation with toxin

[0359] 1. Antibody conjugation

[0360] To the candidate antibodies Clone2-HM, Clone3-HM, Clone5-HM, Clone6-HM, Clone7-HM, 6-fold equivalents of TCEP were added, respectively, and reduced at 25°C for 6 hours, and then 10-fold equivalents of Mal-PEG8-Val-Cit-PAB-MMAE were added to continue coupling for 4 hours; after the coupling was completed, the unreacted Mal-PEG8-VC-PAB-MMAE was removed by using Protein A to bind the antibody; after the antibody was eluted from the affinity chromatography column using glycine at pH 3.0, it was replaced with PBS buffer; and the coupled product was analyzed by using HIC-HPLC to calculate the average DAR value. The positive control antibody Gemtuzumab was coupled with MMAE toxin according to the same scheme.

[0361] 2. In vitro killing of target cells by candidate antibodies coupled with MMAE

[0362] 1). Molm13-Luciferase cells in the logarithmic growth phase were taken, and the cell density was adjusted to 2x10 5 / mL with complete culture medium.

[0363] 2). 100 μL / well of sterile water was added to the four surrounding wells of a 96-well plate; 100 μL / well of cell suspension was added to the remaining wells, which were then placed in a 5% CO2, 37°C incubator for 24 h.

[0364] 3). The candidate antibody and positive antibody coupling products were filtered to remove bacteria through a 0.22 μm filter membrane, and the antibody concentration was adjusted to 20 μg / mL with complete culture medium.

[0365] 4). 240 μL of the first gradient concentration of the mother liquor (antibody concentration of 20 μg / mL) was added to 480 μL of culture medium, and 3-fold gradient dilution was performed, for a total of 9 dilutions. 100 μL of the antibody diluent was added to each well.

[0366] 5). 120 h after the addition of the drug, 100 μL of the cell viability detection kit substrate was added to each cell well, followed by 20 times of blowing, and then the cells were allowed to stand for 10 min. 100 μL of the liquid was transferred from each well to a white non-transparent 96-well plate, and the luminescence value was read by using a Tecan M1000Pro.

[0367] The candidate humanized antibodies coupled with toxins and the positive control antibody can effectively inhibit the proliferation of tumor cells Molm13-Luciferase; and the killing activity of all candidate antibodies coupled with toxins on target cells is stronger than that of the positive control antibody Gemtuzumab (Figure 6).

[0368] 3. In vivo killing of target cells by candidate antibodies coupled with MMAE

[0369] 1). Resuscitate Molm-13-Luciferase cells from liquid nitrogen, adjust to logarithmic growth phase, then adjust the cell density to 5x10 6 / mL with normal saline;

[0370] 2). Take NSG mice, inoculate Molm13-Luciferase cells 5x10 5 / mouse through the tail vein, inoculation volume is 100 μL;

[0371] 3). On the 7th day after tumor inoculation, the mice were subcutaneously injected with 200 μL of 10 mg / mL D-luciferin potassium for the first time, and the mice were injected with different antibody conjugate products (Gemtuzumab-MMAE, Clone3-HM-MMAE) at a dose of 5 mg / kg, and the control group of mice was injected with the same volume of normal saline through the tail vein.

[0372] 4). Subsequent live imaging was performed every 7 days to monitor the proliferation of tumor cells in mice.

[0373] The conjugated toxin candidate humanized antibody Clone3-HM and the positive control antibody can effectively inhibit the proliferation of tumor cells Molm13-Luciferase in mice, and the killing activity of the target cells is stronger than that of the positive control antibody Gemtuzumab (Figure 7), the survival time of the mice is longer (Figure 8), and the body weight of the mice does not decrease significantly during the entire drug 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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