CD33 binding molecule and application thereof

Abstract

The invention relates to a CD33 binding molecule and application thereof. The binding molecule of the CD33 comprises a CDR1, a CDR2 and a CDR3, wherein the CDR1, the CDR2 and the CDR3 are selected from the following amino acid sequences: the CDR1 shown as any one of SEQ ID NO: 24-26; cDR2 as shown in any one of SEQ ID NO: 27-29; the CDR3 is shown as any one of SEQ ID NO: 30-34. According to the invention, the anti-CD33 single-domain antibody specifically combined with CD33 is screened by using a biological gene engineering technology, CDR modification is carried out, and the antibody has good affinity and is used for treating hematological malignant tumors and other CD33 related diseases.

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to a CD33 binding molecule and uses thereof. Background Art

[0002] Acute leukemia is a malignant clonal disease of hematopoietic stem cells. Due to enhanced self-renewal, uncontrolled proliferation, differentiation disorders, and blocked apoptosis of leukemia cells, they stagnate at different stages of cell development. In the bone marrow and other hematopoietic tissues, leukemia cells proliferate and accumulate in large numbers, inhibiting normal hematopoiesis and infiltrating other organs and tissues. Among adult acute leukemia patients, acute myeloid leukemia (AML) is the most common and most fatal type of leukemia. In the past 40 years, although people have a deeper understanding of AML, the treatment of AML has not changed significantly compared with other blood tumors. At present, the cure rate of AML patients aged 60 years and under is 35% to 40%, while the cure rate of AML patients aged over 60 years is only 5% to 15%. Decitabine and azacitidine are a class of small molecule drugs for the treatment of AML that target epigenetic mechanisms. In addition to small molecule drugs, antibody drugs and cell immunotherapy are also important means of targeted therapy.

[0003] CD33 is a 67KD glycosylated transmembrane protein belonging to the Siglec family. It is activated after cross-linking or ligand binding, mediates inhibitory signals, regulates intracellular calcium mobilization, cell adhesion, leukemia cell apoptosis, myeloid cell maturation and cytokine production, etc. During differentiation, CD33 decreases on mature granulocytes but is retained on macrophages, monocytes and dendritic cells. CD33 expression is limited in non-hematopoietic tissues, but is highly expressed in AML cells. In addition, it has been confirmed that CD33 is also expressed in leukemia progenitor cells, making this molecule a very potential therapeutic target for AML. Gentuzumabozogamicin is a human IgG4 CD33 monoclonal antibody coupled to a calicheamicin derivative with a DAR of 2-3 and contains approximately 50% naked antibodies. It was approved by the FDA in September 2017 for newly diagnosed CD33-positive acute myeloid leukemia (AML) in adults, relapsed or refractory AML in adults, and AML in 2-year-old and pediatric patients. IMGN779 (ImmunoGen, Inc., Waltham, Massachusetts) consists of a CD33 monoclonal antibody coupled to indolinobenzodiazeprine pseudodimers (alkylating agents). A clinical study (NCT02674763) was conducted, showing that 50 patients have received IMGN779 with no dose-limiting toxicity (DLT). Adverse events of treatment included febrile neutropenia, epistaxis, nausea, diarrhea, fatigue, abdominal pain, and hypokalemia. Overall, blast counts were reduced in 19 of 24 patients (79%) with measurable circulating blasts, and preliminary results showed safety and efficacy. BI836858 is a fully human IgG1 anti-CD33 antibody that enhances binding to FCγRIIIa to enhance its ADCC activity. Preliminary results from Phase 1b reported the results of 31 patients treated with BI836858, with five patients achieving CR / complete response and another four achieving morphological leukemia-free status. AMG330 (Amgen) is a CD33 / CD3 bispecific T cell engagement (BiTE) antibody structure that directs cytotoxic T cells to CD33-expressing AML cells and kills target cells. It has been shown to be effective in preclinical trials and is currently in Phase 1 clinical trials (NCT02520427). Johnson & Johnson's BiTE JNJ-67371244 is also in Phase 1 clinical trials (NCT03915379). 161533TriKE (GTBiopharma) (CD16 / IL-15 / CD33) enhances natural killer cell function and induces anti-tumor responses targeting CD33 by targeting CD33-positive malignant cells and CD33 myeloid-derived suppressor cells (MDSC).Currently, a phase 1 / 2 clinical study, NCT03214666, GTB-3550 (CD16 / IL-15 / CD33) Tri-Specific Killer Engager (TriKE. TM ) for High Risk Heme Malignancies. AML is a highly malignant blood tumor that highly expresses CD33. Currently, monoclonal antibodies, bispecific antibodies, trispecific antibodies, and ADCs are all developed by companies, and Mylotarg has been approved by the FDA. Although similar to all drug development, there are many difficulties, but CD33 is still the most promising development target for AML. Summary of the invention

[0004] The purpose of the present invention is to provide a binding molecule capable of specifically binding to CD33 and its use.

[0005] The first aspect of the present invention provides a CD33 binding molecule, wherein the CD33 binding molecule is an anti-CD33 single domain antibody, wherein the single domain antibody comprises CDR1, CDR2 and CDR3, and is selected from the following amino acid sequences:

[0006] CDR1 shown in any one of SEQ ID NOs: 24-26;

[0007] CDR2 shown in any one of SEQ ID NOs: 27-29;

[0008] CDR3 shown in any one of SEQ ID NOs: 30-34;

[0009] The above amino acid sequence includes derivative sequences formed by optionally adding, deleting, modifying and / or replacing 1-5 amino acids and capable of retaining CD33 binding affinity.

[0010] Preferably, the amino acid sequences of CDR1, CDR2 and CDR3 are selected from the following groups:

[0011] (1) CDR1 shown in SEQ ID NO:24, CDR2 shown in SEQ ID NO:28, and CDR3 shown in SEQ ID NO:34;

[0012] (2) CDR1 shown in SEQ ID NO:24, CDR2 shown in SEQ ID NO:28, and CDR3 shown in SEQ ID NO:33;

[0013] (3) CDR1 shown in SEQ ID NO:25, CDR2 shown in SEQ ID NO:29, and CDR3 shown in SEQ ID NO:31;

[0014] (4) CDR1 shown in SEQ ID NO:25, CDR2 shown in SEQ ID NO:29, and CDR3 shown in SEQ ID NO:30;

[0015] (5) CDR1 shown in SEQ ID NO:25, CDR2 shown in SEQ ID NO:29, and CDR3 shown in SEQ ID NO:32;

[0016] (6) CDR1 shown in SEQ ID NO:26, CDR2 shown in SEQ ID NO:27, and CDR3 shown in SEQ ID NO:30;

[0017] (7) CDR1 shown in SEQ ID NO:26, CDR2 shown in SEQ ID NO:27, and CDR3 shown in SEQ ID NO:32.

[0018] All of the above sequences can be replaced by sequences having "at least 80% homology" with the sequence or sequences with only one or a few amino acids replaced; preferably "at least 85% homology", more preferably "at least 90% homology", more preferably "at least 95% homology", and most preferably "at least 98% homology".

[0019] Preferably, the single domain antibody consists of heavy chains.

[0020] In one embodiment, one to five arbitrary amino acid residues in any one or more CDRs can be replaced by their conservative amino acids. Specifically, in CDR1, 1 to 5 amino acid residues can be replaced by their conservative amino acids; in CDR2, 1 to 5 amino acid residues can be replaced by their conservative amino acids; in CDR3, 1 to 5 amino acid residues can be replaced by their conservative amino acids.

[0021] As used herein, the term "sequence homology" refers to the extent to which two (nucleotide or amino acid) sequences have identical residues at the same position in an alignment, and is usually expressed as a percentage. Preferably, homology is determined over the entire length of the sequences being compared. Thus, two copies having exactly the same sequence have 100% homology.

[0022] In some embodiments, a sequence that replaces only one or a few amino acids compared to the aforementioned sequence, for example, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions, can also achieve the purpose of the invention. These variant forms include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and optimally 1-10) amino acid deletions, insertions and / or substitutions, and addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. In fact, when determining the degree of sequence homology between two amino acid sequences or when determining the combination of CDR1, CDR2 and CDR3 in a single domain antibody, a technician may consider so-called "conservative" amino acid substitutions, and in the case of substitutions, the substitution will preferably be a conservative amino acid substitution. Conservative amino acids, which can generally be described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue having a similar chemical structure, and the substitution has little or substantially no effect on the function, activity or other biological properties of the polypeptide. Conservative amino acid substitutions are common in the art. For example, conservative amino acid substitutions are substitutions in which one or a few amino acids in the following groups (a)-(d) are replaced by another or a few amino acids in the same group: (a) polar negatively charged residues and their uncharged amides: Asp, Asn, Glu, Gln; (b) polar positively charged residues: His, Arg, Lys; (c) aromatic residues: Phe, Trp, Tyr; (d) aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Gly, Pro, Met, Leu, Ile, Val, Cys. Particularly preferred conservative amino acid substitutions are as follows: Asp is replaced by Glu; Asn is replaced by Gln or His; Glu is replaced by Asp; Gln is replaced by Asn; His is replaced by Asn or Gln; Arg is replaced by Lys; Lys is replaced by Arg, Gln; Phe is replaced by Met, Leu, Tyr; Trp is replaced by Tyr; Tyr is replaced by Phe, Trp; Ala is replaced by Gly or Ser; Ser is replaced by Thr; Thr is replaced by Ser; Gly is replaced by Ala or Pro; Met is replaced by Leu, Tyr or Ile; Leu is replaced by Ile or Val; Ile is replaced by Leu or Val; Val is replaced by Ile or Leu; Cys is replaced by Ser. In addition, those skilled in the art know that the framework region sequences FR1-4 are not immutable, and the sequences of FR1-4 can adopt conservative sequence variants of the sequences disclosed in the present invention.

[0023] The meaning of "anti-CD33 single-domain antibody" in the present invention includes not only complete single-domain antibodies, but also fragments, derivatives and analogs of anti-CD33 single-domain antibodies. As used herein, the terms "fragment", "derivative" and "analog" have the same meaning, and all refer to polypeptides that substantially maintain the same biological function or activity as the antibody of the present invention. The polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substitution group in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusion of an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with an Fc tag). According to the teachings of this article, these fragments, derivatives and analogs belong to the scope known to those skilled in the art.

[0024] In a preferred embodiment, the heavy chain further comprises a framework region FR; the framework region FR comprises the amino acid sequences of FR1, FR2, FR3 and FR4; the framework region FR is selected from the following amino acid sequences:

[0025] FR1 shown in SEQ ID NO:15 or 16;

[0026] FR2 shown in any one of SEQ ID NOs: 17-19;

[0027] FR3 shown in any one of SEQ ID NOs:20-22;

[0028] FR4 shown in SEQ ID NO:23;

[0029] The above amino acid sequence includes derivative sequences formed by optionally adding, deleting, modifying and / or replacing 1-5 amino acids and capable of retaining CD33 binding affinity.

[0030] The second aspect of the present invention is to provide an amino acid sequence of a CD33 binding molecule, wherein the CD33 binding molecule is an anti-CD33 single domain antibody, wherein the amino acid sequence of the single domain antibody is selected from any one of SEQ ID NOs: 1-7, or has at least 80% sequence homology with the amino acid sequence shown in SEQ ID NOs: 1-7, and can specifically bind to the CD33 protein.

[0031] In one embodiment, the anti-CD33 single domain antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology with an amino acid sequence selected from SEQ ID NO: 1-7 or SEQ ID NO: 1-7, and is capable of specifically binding to CD33 protein.

[0032] In one embodiment, the present invention provides an Fc fusion protein or a humanized antibody of a CD33 binding molecule.

[0033] The third aspect of the present invention is to provide a recombinant protein comprising any of the aforementioned CD33 binding molecules.

[0034] In one embodiment, the recombinant protein comprises the aforementioned anti-CD33 single domain antibody.

[0035] Preferably, the recombinant protein may be a single domain antibody as shown in the aforementioned SEQ ID NO: 1-7, or a single domain antibody having at least 80% homology with SEQ ID NO: 1-7, or a multi-epitope antibody, a multi-specific antibody, and a multivalent antibody; for example, a multi-epitope antibody may be composed of more than one sequence in SEQ ID NO: 1-7; a multivalent antibody may be composed of one of the sequences in SEQ ID NO: 1-7 repeated several times; multi-specific antibodies include but are not limited to bispecific antibodies, and tri-specific antibodies; in addition, the recombinant protein may be a fragment, derivative, and analog of the aforementioned antibody.

[0036] The fourth aspect of the present invention is to provide an isolated nucleic acid molecule encoding any of the aforementioned CD33 binding molecules or recombinant proteins.

[0037] Preferably, the nucleotide sequences thereof are shown in SEQ ID NOs: 8-14, respectively, or have at least 95% sequence homology with SEQ ID NOs: 8-14.

[0038] In one embodiment, the nucleic acid molecule encoding the CD33 binding molecule is selected from SEQ ID NOs: 8-14 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology with a nucleotide sequence selected from SEQ ID NOs: 8-14, and the anti-CD33 single domain antibody encoded thereby can specifically bind to the anti-CD33 protein.

[0039] The fifth aspect of the present invention is to provide an expression vector comprising a nucleotide molecule encoding any of the aforementioned CD33 binding molecules, or the nucleotide molecule provided in the fourth aspect.

[0040] Preferably, the expression vector comprises a nucleotide molecule encoding a CD33 binding molecule or an Fc fusion protein or a humanized antibody, and the nucleotide sequence is selected from SEQ ID NOs: 8-14 or has at least 80% sequence homology with any one of SEQ ID NOs: 8-14.

[0041] In a preferred embodiment, the expression vector used is RJK-V4-hFC1 (a nucleotide molecule encoding a CD33 binding molecule or its Fc fusion protein or a humanized antibody is integrated into RJK-V4-hFC1 by genetic engineering means). Other general expression vectors may also be selected as needed.

[0042] The sixth aspect of the present invention provides a host cell, which expresses any of the aforementioned CD33 binding molecules, or comprises the nucleotide molecule provided in the fourth aspect or the expression vector provided in the fifth aspect.

[0043] Preferably, the host cell is capable of expressing the aforementioned CD33 binding molecule, Fc fusion protein or humanized antibody, or contains the aforementioned expression vector.

[0044] In another preferred embodiment, the host cell is a bacterial cell, a fungal cell or a mammalian cell.

[0045] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell, including bacteria or fungi.

[0046] In another preferred embodiment, the host cell is selected from the group consisting of E. coli, yeast cells, mammalian cells, bacteriophages, or a combination thereof.

[0047] In another preferred embodiment, the prokaryotic cell is selected from the group consisting of Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Proteus mirabilis, or a combination thereof.

[0048] In another preferred embodiment, the eukaryotic cell is selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, or a combination thereof.

[0049] In another preferred embodiment, the eukaryotic cell is selected from the group consisting of insect cells such as fall armyworm, plant cells such as tobacco, BHK cells, CHO cells, COS cells, myeloma cells, or a combination thereof.

[0050] In another preferred embodiment, the host cell is a suspension ExpiCHO-S cell.

[0051] In another preferred embodiment, the host cell is a suspension 293F cell.

[0052] The seventh aspect of the present invention is to provide a pharmaceutical composition, comprising the CD33 binding molecule of any of the aforementioned items, the recombinant protein provided by the third aspect, the nucleic acid provided by the fourth aspect, the expression vector provided by the fifth aspect, the host cell provided by the sixth aspect, or a combination thereof; and a pharmaceutically acceptable carrier.

[0053] Preferably, the pharmaceutical composition comprises the aforementioned CD33 binding molecule and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally determined according to the isoelectric point of the antibody (the pH of the aqueous carrier medium needs to deviate from the isoelectric point of the antibody and differ from the isoelectric point of the antibody by about 2).

[0054] The pharmaceutical composition of the present invention can be directly used to bind to anti-CD33 protein molecules.

[0055] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the aforementioned CD33 binding molecule and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions.

[0056] The eighth aspect of the present invention is to provide a medicament for treating a disease, comprising the pharmaceutical composition provided in the seventh aspect.

[0057] Preferably, the aforementioned CD33 binding molecule (single domain antibody for binding to CD33 protein) is contained as an active ingredient.

[0058] The ninth aspect of the present invention is to provide a kit for detecting CD33 levels, comprising any one of the aforementioned CD33 binding molecules (anti-CD33 single domain antibodies).

[0059] In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, a buffer, and the like.

[0060] In a preferred embodiment, the kit includes an antibody that recognizes CD33 protein, a lysis medium for dissolving the sample, and common reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, etc. The detection kit can be an in vitro diagnostic device.

[0061] In a preferred embodiment, the kit further contains a secondary antibody and an enzyme or fluorescent or radioactive label for detection, and a buffer.

[0062] In a preferred embodiment, the second antibody of the kit can be the antibody of the aforementioned anti-CD33 single domain antibody (as anti-antibody), and can be a single domain antibody, a monoclonal antibody, a polyclonal antibody or any other form of antibody.

[0063] The tenth aspect of the present invention provides a method for producing a CD33 binding molecule (anti-CD33 single domain antibody), comprising the steps of:

[0064] (a) culturing the host cell of the sixth aspect of the present invention under conditions suitable for producing the single domain antibody, thereby obtaining a culture containing the anti-CD33 single domain antibody; and

[0065] (b) isolating or recovering the anti-CD33 single domain antibody from the culture; and

[0066] (c) optionally, purifying and / or modifying the anti-CD33 single domain antibody obtained in step (b).

[0067] The eleventh aspect of the present invention is to provide use of the aforementioned CD33 binding molecule (anti-CD33 single domain antibody) or the aforementioned pharmaceutical composition in the preparation of a drug for treating tumor diseases.

[0068] In a preferred embodiment, the disease is a hematological tumor.

[0069] In a preferred embodiment, the disease is acute myeloid leukemia.

[0070] The twelfth aspect of the present invention is to provide a method for treating a disease using the aforementioned CD33 binding molecule (anti-CD33 single domain antibody) or the aforementioned pharmaceutical composition.

[0071] In a preferred embodiment, the disease is a hematological tumor.

[0072] In a preferred embodiment, the disease is acute myeloid leukemia.

[0073] The present invention is beneficial in that:

[0074] Compared with the prior art, the beneficial effects of the present invention are:

[0075] (1) The single domain antibody of the present invention is specific to the anti-CD33 protein with the correct spatial structure.

[0076] (2) The single-domain antibody obtained in the present invention has a flexible expression system selection, and can be expressed in a prokaryotic system or a eukaryotic system of yeast cells or mammalian cells. In addition, the expression cost in the prokaryotic expression system is low, which can reduce the later production cost.

[0077] (3) The single-domain antibodies obtained by the present invention are easy to modify in multiple combinations. Multivalent and multispecific antibodies can be obtained by simple concatenation through genetic engineering. In addition, their immune heterogeneity is very low and they will not produce a strong immune response without humanization.

[0078] (4) The single domain antibody obtained by the present invention has a wider affinity range. Before affinity maturation, its affinity range can range from nM level to pM level, providing multiple options for antibodies with different uses in the later stage. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] In order to more clearly illustrate the technical solution of the present application, the drawings required for use in the embodiments are briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0080] Figure 1 The antibody-antigen binding dose-effect curve measurement profile (1B1-V2, 1B1-V3) of Example 12;

[0081] Figure 2 The antibody-antigen binding dose-effect curve of Example 12 (1B10-V3, 1D7);

[0082] Figure 3 The antibody-antigen binding dose-effect curve measurement spectrum (1D7-V2) of Example 12;

[0083] Figure 4 The antibody-antigen binding dose-effect curve measurement spectrum (1B10-V1) of Example 12;

[0084] Figure 5 The ADCC effect determination profiles of the antibodies in Example 13 (1B1-V2, 1B1-V3);

[0085] Figure 6 The ADCC effect determination profiles of the antibodies in Example 13 (1D7-V2, 1D7-V3);

[0086] Figure 7 The ADCC effect determination profile of the antibody in Example 13 (hIgG, Tab3);

[0087] Figure 8The ADCC effect determination spectrum (1D7) of the antibody in Example 13;

[0088] Fig. 9 The ADCC effect determination spectrum of the antibody in Example 13 (1B10-V3);

[0089] Fig.10 This is the ADCC effect measurement graph of the antibody in Example 13 (1B10-V1). DETAILED DESCRIPTION

[0090] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0091] As used herein, "single domain antibodies" (sdAb, also referred to as nanobodies or VHHs by developer Ablynx) are well known to those skilled in the art. Single domain antibodies are antibodies whose complementary determining regions are part of a single domain polypeptide. Thus, single domain antibodies comprise a single complementary determining region (a single CDR1, a single CDR2, and a single CDR3). Examples of single domain antibodies are antibodies with only heavy chains (which naturally do not comprise light chains), single domain antibodies derived from conventional antibodies, and engineered antibodies.

[0092] Single domain antibodies can be derived from any species, including mice, humans, camels, llamas, goats, rabbits and cattle. For example, naturally occurring VHH molecules can be derived from antibodies provided by camelid species (e.g., camels, dromedaries, llamas and guanacos). Like complete antibodies, single domain antibodies are able to selectively bind to specific antigens. Single domain antibodies can contain only the variable domains of immunoglobulin chains, which have CDR1, CDR2 and CDR3 and framework regions.

[0093] As used herein, the term "Fc fusion protein" refers to a novel protein produced by fusing the Fc segment of a target antibody with a functional protein molecule having biological activity using genetic engineering technology.

[0094] The term "humanized antibody" refers to an antibody obtained by fusing the heavy chain variable region of a target antibody (such as an animal antibody) with the constant region of a human antibody, or an antibody obtained by transplanting the complementary determining region (CDR1-3) of a target antibody into the variable region of a human antibody, or an antibody obtained by mutating the amino acids of the target antibody according to the characteristics of the human antibody framework region (FR1-4). Humanized antibodies can be synthesized or by site-directed mutagenesis.

[0095] In the present invention, sequences with high sequence homology to the CDR1-3 disclosed in the present invention can also obtain single domain antibodies against CD33. In some embodiments, sequences with "at least 80% homology" or "at least 85% homology", "at least 90% homology", "at least 95% homology", "at least 98% homology" to the sequences in SEQ ID NO: 1-7 can achieve the purpose of the invention.

[0096] In some embodiments, sequences that replace only one or a few amino acids compared to the sequences in SEQ ID NO: 1-7, for example, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions, can also achieve the purpose of the invention. In fact, when determining the degree of sequence homology between two amino acid sequences or when determining the combination of CDR1, CDR2 and CDR3 in a single domain antibody, a technician may consider so-called "conservative" amino acid substitutions. In the case of substitutions, the substitution will preferably be a conservative amino acid substitution, which can generally be described as an amino acid substitution in which an amino acid residue is replaced by another amino acid residue with a similar chemical structure, and the substitution has little or substantially no effect on the function, activity or other biological properties of the polypeptide. Conservative amino acid substitutions are common in the art. For example, conservative amino acid substitutions are substitutions in which one or a few amino acids in the following groups (a)-(d) are replaced by another or a few amino acids in the same group: (a) polar negatively charged residues and their uncharged amides: Asp, Asn, Glu, Gln; (b) polar positively charged residues: His, Arg, Lys; (c) aromatic residues: Phe, Trp, Tyr; (d) aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Gly, Pro, Met, Leu, Ile, Val, Cys. Particularly preferred conservative amino acid substitutions are as follows: Asp is replaced by Glu; Asn is replaced by Gln or His; Glu is replaced by Asp; Gln is replaced by Asn; His is replaced by Asn or Gln; Arg is replaced by Lys; Lys is replaced by Arg, Gln; Phe is replaced by Met, Leu, Tyr; Trp is replaced by Tyr; Tyr is replaced by Phe, Trp; Ala is replaced by Gly or Ser; Ser is replaced by Thr; Thr is replaced by Ser; Gly is replaced by Ala or Pro; Met is replaced by Leu, Tyr or Ile; Leu is replaced by Ile or Val; Ile is replaced by Leu or Val; Val is replaced by Ile or Leu; Cys is replaced by Ser. Preferred host cells of the present invention are bacterial cells, fungal cells or mammalian cells.

[0097] Antibody-dependent cell-mediated cytotoxicity (ADCC) refers to the killing of target cells by NK cells, macrophages, and neutrophils that express IgGFc receptors by binding to the Fc segment of IgG antibodies that have bound to the surface of target cells such as virus-infected cells and tumor cells.

[0098] This patent is to prepare the target protein and the truncated form of the target protein through genetic engineering technology, and then immunize the Alxa Bactrian camel in Inner Mongolia with the obtained antigen protein. After multiple immunizations, the peripheral blood lymphocytes or spleen cells of the camel are obtained. The variable region coding sequence of the camel-derived antibody is recombined into the phage display vector through genetic engineering. The specific antibodies against the antigen protein are screened through phage display technology, and their ability to bind to the antigen and their application in inhibiting tumors, autoimmunity and inflammatory diseases are further tested.

[0099] The above technical solution is now broken down and explained in detail in the form of specific embodiments:

[0100] Example 1: Preparation of human CD33 protein:

[0101] The human recombinant extracellular domain protein used in this patent is obtained by the company's own expression and purification. The expression vector design scheme of the human recombinant CD33 protein is as follows:

[0102] (1) The coding sequence of CD33 was retrieved from NCBI and its accession number is NM_001772.3. The accession number of the amino acid sequence encoded by this sequence is NP_001763.3.

[0103] (2) The nucleotide sequence encoding the 1st to 259th amino acids of the CD33 protein was cloned into the vector pcDNA3.4-CH by gene synthesis. The constructed vector was subjected to Sanger sequencing and compared with the original sequence. After confirmation, the recombinant plasmid was extracted in large quantities to remove endotoxins and transfected into suspended 293F cells for expression and purification of the target protein. The purity reached more than 90%, meeting the requirements for animal immunization.

[0104] Example 2: Construction of a single domain antibody library against CD33 protein:

[0105] 1 mg of the human recombinant CD33 protein purified in Example 1 was mixed with an equal volume of Freund's complete adjuvant to immunize a Bactrian camel from Alxa, Inner Mongolia, once a week for a total of 7 consecutive immunizations. For the first immunization, the adjuvant used was "Freund's complete adjuvant", the immunization dose should be twice the subsequent immunization dose, and the interval between the first immunization and the second immunization was 10 days. Except for the first immunization, the remaining six immunizations were all performed by mixing 1 mg of CD33 protein with an equal volume of Freund's incomplete adjuvant for animal immunization. The immunization process is to centrally stimulate the camel to produce antibodies against the CD33 protein.

[0106] After the animal immunization, 150 mL of peripheral blood lymphocytes were extracted from the camel, and RNA was extracted from the cells. The extracted total RNA was used to synthesize cDNA, and VHH (antibody heavy chain variable region) was amplified using cDNA as a template through a nested PCR reaction.

[0107] Then, the pMECS vector and VHH fragment were digested with restriction endonucleases, and the digested fragments and vector were linked. The linked fragments were electroporated into competent cells TG1 to construct a phage display library of CD33 protein and measure the library capacity. The library capacity was about 1×10 9 At the same time, the correct insertion rate of the library in the target fragment was detected by colony PCR identification.

[0108] The results showed that after PCR amplification of 30 colonies randomly selected from the library, 28 clones could amplify bands of the predicted size, and 2 clones amplified incorrect bands, so the correct insertion rate was 28÷30×100%≈93.3%.

[0109] Example 3: Screening of single domain antibodies against CD33 protein:

[0110] 200 μL of the recombinant TG1 cells in Example 2 were cultured in 2×TY medium, during which 40 μL of helper phage VCSM13 was added to infect the TG1 cells and cultured overnight to amplify the phages. The next day, the phages were precipitated with PEG / NaCl and the amplified phages were collected by centrifugation.

[0111] 500 μg of CD33 protein diluted in 100 mM NaHCO3 at pH 8.3 was coupled to an ELISA plate and placed at 4°C overnight. A negative control well (culture medium control) was set up at the same time. On the second day, 200 μL of 3% skim milk was added and the plate was blocked at room temperature for 2 hours. After the blocking, 100 μL of amplified phage library (approximately 2×10 11 After 1 hour, the cells were washed 15 times with PBS + 0.05% Tween-20 to remove unbound phages.

[0112] The phages specifically bound to the CD33 protein were dissociated with trypsin at a final concentration of 25 mg / mL and infected with Escherichia coli TG1 cells in the logarithmic growth phase. The cells were cultured at 37°C for 1 hour to produce and collect phages for the next round of screening. The same screening process was repeated for one round to gradually achieve enrichment.

[0113] Example 4: Screening of specific positive clones against CD33 using phage enzyme-linked immunosorbent assay (ELISA):

[0114] According to the screening method in Example 3, two rounds of screening were performed for the single domain antibody against CD33 protein, and the phage enrichment factor of the anti-CD33 protein reached more than 10. After the screening, 384 single colonies were selected from the positive clones obtained in the screening and inoculated into 96 deep-well plates in 2×TY medium containing 100 μg / mL ampicillin, and a blank control was set. After culturing at 37° C. to the logarithmic phase, IPTG with a final concentration of 1 mM was added, and the plates were cultured at 28° C. overnight.

[0115] The crude antibody was obtained by osmotic swelling method; CD33 recombinant protein was released into 100mM NaHCO3 at pH 8.3 and 100μg protein was coated in an ELISA plate at 4°C overnight. 100μL of the crude antibody extract was transferred to the ELISA plate with antigen added and incubated at room temperature for 1 hour; unbound antibody was washed with PBST, and 100μl of Mouse Anti-HA tag Antibody (HRP) (mouse anti-HA horseradish peroxidase labeled antibody, ThermoFisher) diluted 1:2000 was added and incubated at room temperature for 1 hour; unbound antibody was washed with PBST, and horseradish peroxidase colorimetric solution was added. After reacting at 37°C for 15 minutes, stop solution was added and the absorbance value was read at a wavelength of 450nm on the microplate reader.

[0116] When the OD value of the sample well is more than 5 times greater than that of the control well, it is determined to be a positive clone well; the bacteria in the positive clone well are transferred and shaken in LB medium containing 100 μg / mL ampicillin to extract the plasmid and perform sequencing.

[0117] The gene sequences of each clone were analyzed using the sequence alignment software VectorNTI. Strains with identical CDR1, CDR2, and CDR3 sequences were considered the same clone, while strains with different sequences were considered different clones. Ultimately, single-domain antibodies specific for CD33 protein were obtained (including 1B10, 1D7, and 1B1).

[0118] The amino acid sequence of the antibody is a FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 structure, which constitutes the variable domain (VHH) of the entire heavy chain antibody. The obtained single domain antibody recombinant plasmid can be expressed in a prokaryotic system to ultimately obtain a single domain antibody protein.

[0119] The CDR3 of 1D7 was modified to obtain single-domain antibodies 1D7-V2 and 1D7-V3. The CDR3 of 1B1 was modified to obtain single-domain antibodies 1B1-V2 and 1B1-V3. The CDR3 and CDR1 of 1B10 were modified to obtain single-domain antibodies 1B10-V1 and 1B10-V3.

[0120] The amino acid sequences of the single domain antibodies 1B1-V2, 1B1-V3, 1B10-V1, 1B10-V3, 1D7, 1D7-V2 and 1D7-V3 are shown in SEQ ID NOs: 1-7, respectively, and the nucleotide sequences thereof are shown in SEQ ID NOs: 8-14, respectively.

[0121] The CDR sequences of the seven single domain antibodies are shown in Tables 1, 2, and 3, the FR sequences of the seven single domain antibodies are shown in Tables 4, 5, 6, and 7, and the protein sequences and amino acid sequences of the seven single domain antibodies are shown in Tables 8 and 9, respectively.

[0122] The CDR sequences of 1B1 and 1B10 are shown in Table 10, CDR1-3 of 1B1 corresponds to SEQ ID NOs: 35-37, CDR1-3 of 1B10 corresponds to SEQ ID NOs: 38-40, and the full-length amino acid sequences of 1B1 and 1B10 are SEQ ID NOs: 41-42, respectively (Table 11).

[0123] Table 1: CDR1 sequences of 7 single domain antibodies

[0124]

[0125] Table 2: CDR2 sequences of 7 single domain antibodies

[0126]

[0127] Table 3: CDR3 sequences of 7 single domain antibodies

[0128]

[0129] Table 4: FR1 sequences of 7 single domain antibodies

[0130]

[0131] Table 5: FR2 sequences of 7 single domain antibodies

[0132]

[0133] Table 6: FR3 sequences of 7 single domain antibodies

[0134]

[0135] Table 7: FR4 sequences of 7 single domain antibodies

[0136]

[0137] Table 8: Amino acid sequences of 7 single domain antibodies

[0138]

[0139]

[0140] Table 9: Nucleotide sequences of 7 single domain antibodies

[0141]

[0142]

[0143] Table 10 CDR sequences of 1B1 and 1B10

[0144]

[0145] Table 11 Full-length amino acid sequences of 1B1 and 1B10

[0146]

[0147] Example 5: Purification and expression of specific single domain antibodies against CD33 protein in host bacteria Escherichia coli

[0148] The plasmids (pMECS-VHH) of different clones obtained by sequencing analysis in Example 4 were electrotransformed into Escherichia coli HB2151, and spread on LB+amp+glucose, i.e., culture plates containing ampicillin and glucose, and cultured at 37°C overnight; a single colony was selected and inoculated into 5 mL of LB culture medium containing ampicillin, and cultured at 37°C in a shaking incubator overnight.

[0149] Inoculate 1 mL of overnight cultured bacteria into 330 mL of TB culture medium, culture at 37°C in a shaking incubator, add 1 M IPTG when the OD600nm value reaches 0.6-0.9, and culture overnight in a shaking incubator at 28°C; centrifuge to collect E. coli, and use the osmotic swelling method to obtain a crude antibody extract;

[0150] The antibody was purified by nickel column affinity chromatography.

[0151] Example 6: Construction of a eukaryotic expression vector for Fc fusion antibody of an anti-CD33 single domain antibody

[0152] (1) Subcloning the target sequence obtained in Example 4 into a eukaryotic expression vector: The antibody screened in Example 4 was sequenced by Sanger sequencing to obtain its nucleotide sequence;

[0153] (2) synthesizing the above nucleotide sequences (SEQ ID NOs: 8-14 and nucleotide sequences of other single-domain antibody clones whose sequences are not shown) into the vector RJK-V4-hFC1 designed and modified by our company to obtain a recombinant eukaryotic expression vector. The modification method of the vector is as described in Example 10;

[0154] (3) transforming the recombinant eukaryotic expression vector constructed in step (2) into DH5α Escherichia coli, culturing for plasmid extraction, and removing endotoxin;

[0155] (4) Sequencing the extracted plasmid for further identification;

[0156] (5) The confirmed recombinant vector is prepared for subsequent eukaryotic cell transfection and expression, and the Fc protein of VHH is expressed by the method of Example 7 or 8, and the above antibody is purified by the method of Example 9.

[0157] Example 7: Expression of single domain antibodies against CD33 protein in suspension ExpiCHO-S cells

[0158] (1) 3 days before transfection, 2.5×10 5 / mL cell passaging and expansion of ExpiCHO-S TM Transfer the calculated volume of cells to a fresh 120 mL (final volume) of pre-warmed ExpiCHO TM The cell concentration was about 4 × 10 6 -6×10 6 Viable cells / mL;

[0159] (2) One day before transfection, place ExpiCHO-S TM The cells were diluted to a concentration of 3.5 × 10 6 viable cells / mL, cells were cultured overnight;

[0160] (3) On the day of transfection, measure the cell density and percentage of viable cells. The cell density should reach about 7×10 6 -10×10 6 Viable cells / mL;

[0161] (4) Use fresh ExpiCHO preheated to 37°C TMDilute the cells to 6 × 10 6 The calculated required volume of cells was transferred to a fresh 100 mL (final volume) of pre-warmed ExpiCHO TM in a 500 mL shake flask containing expression medium;

[0162] (5) Gently invert to mix ExpiFectamine TM CHO reagent, 3.7 mL OptiPRO TM Dilute ExpiFectamine in culture medium TM CHO reagent, swirl or mix;

[0163] (6) Use 4 mL of Refrigerated OptiPRO TM The plasmid DNA (the plasmid DNA is the Fc fusion antibody eukaryotic expression vector of the anti-CD33 single domain antibody prepared in Example 6) was diluted with the culture medium and mixed by vortexing;

[0164] (7) Incubate the ExpiFectamine CHO / plasmid DNA complex at room temperature for 1-5 minutes, then gently add it to the prepared cell suspension, gently swirling the flask during the addition process;

[0165] (8) The cells were cultured with shaking at 37°C, 8% CO2, and humidified air;

[0166] (9) On the first day after transfection (18-22 hours later), add 600 μl of ExpiFectamine TM CHO Enhancer and 24mLExpiCHO feed;

[0167] (10) Collect the supernatant about 8 days after transfection (cell viability is less than 70%).

[0168] Example 8: Expression of single domain antibodies against CD33 protein in suspension 293F cells

[0169] Recombinant single domain antibody expression experimental process (taking 500mL shake flask as an example):

[0170] (1) 3 days before transfection, 2.5×10 5 / mL cell passage and expansion culture of 293F cells, the calculated required cell volume was transferred to a 500mL shake flask filled with fresh pre-warmed 120mL (final volume) of OPM-293CD05 Medium. The cell concentration reached about 2×10 6 -3×10 6 Viable cells / mL.

[0171] (2) On the day of transfection, measure the cell density and percentage of viable cells. The cell density should reach about 2×10 6 -3×10 6 Viable cells / mL.

[0172] (3) Dilute the cells to 1×10 using pre-warmed OPM-293CD05 Medium. 6 The calculated volume of cells was transferred to a 500 mL shake flask containing 100 mL (final volume) of fresh pre-warmed culture medium.

[0173] (4) Dilute PEI (1 mg / mL) reagent with 4 mL Opti-MEM medium, vortex or pipette to mix; dilute plasmid DNA (plasmid DNA is the Fc fusion antibody eukaryotic expression vector of the anti-CD33 single domain antibody prepared in Example 6) with 4 mL Opti-MEM medium, vortex to mix, and filter with a 0.22 um filter. Incubate at room temperature for 5 minutes.

[0174] (5) Add the diluted PEI reagent to the diluted DNA and mix by inverting. Incubate the PEI / plasmid DNA complex at room temperature for 15-20 minutes, then gently add it to the prepared cell suspension, gently swirling the flask during the addition process.

[0175] (6) The cells were cultured at 37°C, 5% CO2, and shaking at 120 rpm.

[0176] (7) Add 5 mL of OPM-CHO PFF05 feed at 24 and 72 hours after transfection.

[0177] (8) Collect the supernatant approximately 7 days after transfection (when cell viability is less than 70%).

[0178] Example 9: Purification of single domain antibodies against CD33 protein

[0179] (1) The protein expression supernatant obtained in Example 7 or 8 was filtered through a 0.45 μm disposable filter to remove insoluble impurities;

[0180] (2) Purifying the filtrate by affinity chromatography using a protein purifier, utilizing the ability of human Fc to bind to Protein A, and using agarose filler coupled to Protein A for purification;

[0181] (3) The filtrate is passed through the Protein A prepacked column at a flow rate of 1 mL / min. In this step, the target protein in the filtrate will bind to the filler;

[0182] (4) washing the impure proteins bound to the column with low salt (1× PBS) and high salt buffer (10× PBS);

[0183] (5) Elute the target protein bound to the column using glycine-HCl buffer at pH 3.0;

[0184] (6) The eluate was quickly added with a Tris-HCl solution at pH 9.0 to neutralize it;

[0185] (7) The neutralized protein solution is dialyzed and then subjected to SDS-PAGE analysis to determine that the protein purity is above 95% and the concentration is above 0.5 mg / mL, and then stored at low temperature for future use.

[0186] Example 10: Construction of single domain antibody eukaryotic expression vector

[0187] The single-domain antibody universal target vector RJK-V4-hFC1 used in this example is a modification of our company based on the invitrogen commercial vector pCDNA3.4 by fusing the Fc segment in the heavy chain coding sequence of human IgG1, that is, the vector contains the hinge region (Hinge) CH2 and CH3 region of the IgG1 heavy chain. The specific modification plan is as follows:

[0188] (1) Select the restriction enzyme sites XbaI and AgeI on pcDNA3.4;

[0189] (2) introducing a multiple cloning site (MCS) and a 6×His tag at the 5′ and 3′ ends of the Fc fragment coding sequence respectively by overlapping PCR;

[0190] (3) amplifying the above fragment by PCR using a pair of primers with XbaI and AgeI restriction sites respectively;

[0191] (4) using restriction endonucleases XbaI and AgeI to digest pcDNA3.4 and the recombinant DNA fragments in (3), respectively;

[0192] (5) The digested vector and the inserted fragment are ligated with T4 ligase, and the ligated product is then transformed into Escherichia coli, amplified, and sequenced to obtain a recombinant plasmid.

[0193] Example 11: Expression and purification of tool antibody (Tab) targeting human CD33

[0194] Tab3 used in Examples 12-14 of the present invention is lintuzumab. Its sequence was searched in IMGT, and the sequence was entrusted to General Biosystems (Anhui) Co., Ltd. for mammalian cell expression system codon optimization and cloned into pcDNA3.1 vector. After resistance screening, plasmid-positive bacteria were selected for amplification, and plasmids were extracted using a plasmid extraction kit (Macherey Nagel, Cat#740412.50). 100 μg of plasmid (40 μg heavy chain + 60 μg light chain) was added per 100 mL of cells, and transient expression was performed in 293F cells (culture medium: FreeStyle 293 Expression medium, Thermo, Cat#12338026+F-68, Thermo, Cat#24040032) using PEI; 5% volume of 10% Peptone (Sigma, Cat#P0521-100G) was added 6 to 24 hours after transfection, and cultured for about 7 to 8 days at 8% CO2 and 130rpm; when the cell viability dropped to 50%, the expression supernatant was collected and purified using a ProteinA (GE, Cat#17-5438-02) gravity column; after dialysis against PBS, the concentration was determined using Nanodrop, the purity was identified by SEC, and the binding ability was verified by indirect ELISA.

[0195] The Tab3 obtained by the method has a concentration of not less than 2 mg / ml and a purity of more than 95%.

[0196] Example 12: Antibody antigen binding dose-effect curve determination

[0197] In this example, the antigen binding dose-effect curve of the antibody was determined using a standard enzyme-linked immunosorbent assay (ELISA) procedure. The steps are as follows:

[0198] (1) Coat with 50 μL of 1 μg / mL CD33 protein (CD33 protein purchased from Beijing Biopsies Biotechnology Co., Ltd., Catalog #CD3-H5226) at 4°C overnight.

[0199] (2) Wash the plate; add 200 μL of 5% milk and block at 37°C for 2 hours.

[0200] (3) Dilute VHH-hFc to 2ug / mL, and then dilute the antibody 5-fold gradient to a total of 8 concentration gradients. The VHH-hFc here is obtained by purifying the Fc fusion antibody of the single domain antibody against CD33 protein prepared in Example 8 (expressed in 293F cells) by Example 9. In addition, hIgG and Tab controls are set respectively; Tab is prepared in Example 11; wherein hIgG refers to an isotype control, an immunoglobulin molecule that does not bind to any target, and is purchased commercially, and the same is true below.

[0201] (4) Wash the plate; add 50 μL of the single domain antibody diluted in step (3), duplicate wells, and incubate at 37° C. for 1 hour.

[0202] (5) Wash the plate; add 50 μL of HRP-Goat anti hIgG secondary antibody and incubate at 37°C for 30 min.

[0203] (6) Wash the plate; add 50 μL of TMB that has been restored to room temperature and react at room temperature for 15 minutes in the dark.

[0204] (7) Add 50 μL of stop solution (1N HCl) and read the result with a microplate reader.

[0205] (8) Draw the curve and calculate EC 50 ,like Figure 1-Figure 4 The sequences of the single domain antibodies 1D7-V4, 1D7-V5, 1B10-V2, 1B6, 1B10-V4, and 1D7-V1 are not shown.

[0206] from Figure 1-4 It can be seen that the single-domain antibodies 1B1-V2, 1B1-V3, 1B10-V1, 1B10-V3, 1D7, and 1D7-V2 of the present invention all have strong specificity and affinity for CD33 protein.

[0207] Example 13: Determination of ADCC effect of antibodies

[0208] The ADCC effect of antibodies was determined using the reporter gene method. The steps are as follows:

[0209] (1) The cells (CHO-K1-CD33) of passage 3-4 after recovery were collected and plated into 96-well plates at 10,000 cells / well. The method for constructing the tumor cell (CHO-K1-CD33) cell line is as shown in Example 14;

[0210] (2) Tab3 and antibody samples were prepared into a solution with a maximum concentration of 10 μg / mL, and then diluted 10 times in a gradient to obtain 7 concentrations; the antibody sample here refers to the Fc fusion protein of the single domain antibody prepared in Example 8 (expressed in 293F cells) and purified by the method of Example 9. Tab3 and Tab4 were prepared by the method of Example 11.

[0211] (3) adding the gradient diluted antibody solution to the cell culture wells in equal volumes of the cell suspension;

[0212] (4) For the sample wells and E / T wells (antibody concentration is 0), Jurkat-NFAT-luc-FcγRIIIa cells were collected and added to the cell culture wells at a rate of 30,000 cells per well;

[0213] (5) After 6 hours of incubation, cell killing was detected using the One-Glo kit and luminescence was read;

[0214] (6) Calculate the fold of induction = (sample-BG) / (E / T-BG);

[0215] According to the target cell killing rate and concentration, four-parameter fitting was performed to calculate the EC of ADCC mediated by each antibody. 50 Concentration. Figure 5-Figure 10 The sequences of the single-domain antibodies 1B1-V1, 1B1-V4, 1B10-V4, 1D7-V1, 1B1-V5, 3C12, 1B6-V1, 1B10-V2, and 1B6 are not shown.

[0216] Depend on Figure 5-Figure 10 It can be seen that the EC50 values ​​of the single domain antibodies 1B1-V2, 1B1-V3, 1B10-V1, 1B10-V3, 1D7, 1D7-V2 and 1D7-V3 of the present invention are comparable to the EC50 values ​​of Tab3 and Tab4, indicating that they have excellent ADCC effects.

[0217] Example 14: Construction of CHO-K1-CD33 cell line

[0218] The CDS region sequence information of CD33 (accession number: NP_001763.3) was obtained from the NCBI database, and the DNA sequence was selected by gene synthesis and cloned into the PHB-Puro expression vector to construct the PHB-CD33-Puro lentiviral vector plasmid and lentiviral packaging. The CMV-CD33-EF1a-Puromycin gene was introduced into the CHO-K1 genome by the lentiviral infection method, and the CHO-K1-CD33 stable cell line stably expressing CD33 membrane protein was obtained by Puromycin resistance screening and monoclonal screening.

[0219] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A CD33 binding molecule, characterized in that: The CD33 binding molecule is a single domain antibody against CD33, wherein the single domain antibody comprises CDR1, CDR2 and CDR3, wherein the CDR1, CDR2 and CDR3 are selected from the following amino acid sequences: CDR1 shown in any one of SEQ ID NOs: 24-26; CDR2 shown in any one of SEQ ID NOs: 27-29; CDR3 shown in any one of SEQ ID NOs: 30-34; The amino acid sequence includes a derivative sequence formed by optionally adding, deleting, modifying and / or replacing 1 to 5 amino acids and capable of retaining CD33 binding affinity.

2. A CD33 binding molecule according to claim 1, characterized in that: The amino acid sequences of the CDR1, CDR2 and CDR3 are selected from the following groups: (1) CDR1 shown in SEQ ID NO:24, CDR2 shown in SEQ ID NO:28, and CDR3 shown in SEQ ID NO:34; (2) CDR1 shown in SEQ ID NO:24, CDR2 shown in SEQ ID NO:28, and CDR3 shown in SEQ ID NO:33; (3) CDR1 shown in SEQ ID NO:25, CDR2 shown in SEQ ID NO:29, and CDR3 shown in SEQ ID NO:31; (4) CDR1 shown in SEQ ID NO:25, CDR2 shown in SEQ ID NO:29, and CDR3 shown in SEQ ID NO:30; (5) CDR1 shown in SEQ ID NO:25, CDR2 shown in SEQ ID NO:29, and CDR3 shown in SEQ ID NO:32; (6) CDR1 shown in SEQ ID NO:26, CDR2 shown in SEQ ID NO:27, and CDR3 shown in SEQ ID NO:30; (7) CDR1 shown in SEQ ID NO:26, CDR2 shown in SEQ ID NO:27, and CDR3 shown in SEQ ID NO:

32.

3. A CD33 binding molecule according to claim 1, characterized in that: The single domain antibody is composed of heavy chains.

4. A CD33 binding molecule according to claim 3, characterized in that: The heavy chain further comprises a framework region FR; the framework region FR comprises the amino acid sequences of FR1, FR2, FR3 and FR4; the framework region FR is selected from the following amino acid sequences: FR1 shown in SEQ ID NO:15 or 16; FR2 shown in any one of SEQ ID NOs: 17-19; FR3 shown in any one of SEQ ID NOs:20-22; FR4 shown in SEQ ID NO:23; The amino acid sequence includes a derivative sequence formed by optionally adding, deleting, modifying and / or replacing 1 to 5 amino acids and capable of retaining CD33 binding affinity.

5. The CD33 binding molecule according to claim 1, characterized in that: The amino acid sequence of the single-domain antibody is selected from any one of SEQ ID NOs: 1-7, or has at least 80% sequence homology with any one of SEQ ID NOs: 1-7. The humanized antibody or Fc fusion protein of the CD33 binding molecule according to any one of claims 1 to 5.

7. A recombinant protein, characterized in that: The recombinant protein comprises the CD33 binding molecule according to any one of claims 1 to 5.

8. An isolated nucleic acid, characterized in that: The nucleic acid encodes the CD33 binding molecule according to any one of claims 1 to 5, or the recombinant protein according to claim 7.

9. The isolated nucleic acid according to claim 8, characterized in that: The nucleic acid sequence is selected from any one of SEQ ID NOs: 8-14, or has at least 80% sequence homology with any one of SEQ ID NOs: 8-14.

10. An expression vector, characterized in that: It comprises a nucleotide molecule encoding a CD33 binding molecule according to any one of claims 1 to 5 or a nucleic acid according to claim 8 or 9.

11. A host cell, characterized in that: It expresses the CD33 binding molecule according to any one of claims 1 to 5, or comprises the nucleic acid according to claims 8 to 9, or comprises the expression vector according to claim 10.

12. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises a CD33 binding molecule selected from any one of claims 1 to 5, a recombinant protein of claim 7, a nucleic acid of claim 8 or 9, an expression vector of claim 10, a host cell of claim 11, or a combination thereof; and a pharmaceutically acceptable carrier.

13. Use of the CD33 binding molecule according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 12 in the preparation of a drug for treating a disease.

14. A method for treating tumor diseases using the CD33 binding molecule according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 12.

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