Anti-CD33 single-domain antibody, Anti-CD123 antibody, polypeptide targeting CD33 and CD123 molecules, and use thereof

Abstract

An anti-CD33 single-domain antibody, an anti-CD123 antibody, a polypeptide targeting CD33 and CD123 molecules, and use thereof. Using biological genetic engineering technology, the anti-CD33 single-domain antibody specifically binding to CD33, the anti-CD123 single-domain antibody specifically binding to CD123, the polypeptide targeting CD123 and CD33, and the use thereof are selected. The antibodies have good affinity and can be used for treating hematological malignancies and other diseases related to CD123 or CD33.

Description

An anti-CD33 single-domain antibody, an anti-CD123 antibody, a polypeptide targeting CD33 and CD123 molecules, and uses thereof

[0001] Related applications

[0002] This application claims priority to Chinese patent applications filed on February 5, 2024, with application number 2024101671473, entitled “A single-domain antibody against CD33 and its use”, application number 2024101668131, entitled “An anti-CD123 antibody and its use”, and application number 2024101669581, entitled “A polypeptide, composition and its use targeting CD33 and CD123 molecules”, the entire text of which is hereby incorporated by reference. Technical Field

[0003] The present invention relates to the field of antibody technology, and in particular to an anti-CD33 single-domain antibody, an anti-CD123 antibody, a polypeptide targeting CD33 and CD123 molecules, and uses thereof. Background Art

[0004] Acute leukemia is a malignant clonal disease of hematopoietic stem cells. Leukemic cells are arrested at various stages of development due to enhanced self-renewal, uncontrolled proliferation, impaired differentiation, and blocked apoptosis. Leukemic cells proliferate and accumulate in the bone marrow and other hematopoietic tissues, suppressing normal hematopoiesis and infiltrating other organs and tissues. Among adult acute leukemias, acute myeloid leukemia (AML) is the most common and most fatal type of leukemia. Despite a deeper understanding of AML over the past 40 years, its treatment has remained largely unchanged compared to other hematologic malignancies. Currently, the cure rate for AML patients aged 60 years and younger is 35% to 40%, while for those aged 60 years and older, the cure rate is only 5% to 15%. Decitabine and azacitidine are small molecule drugs used to treat AML that target epigenetic mechanisms. In addition to small molecule drugs, antibody-based drugs and cellular immunotherapy are also important targeted therapies.

[0005] CD33 is a 67-kDa glycosylated transmembrane protein belonging to the Siglec family. Activated upon cross-linking or ligand binding, it mediates inhibitory signals, regulating intracellular calcium mobilization, cell adhesion, leukemic cell apoptosis, myeloid cell maturation, and cytokine production. During differentiation, CD33 expression 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. Furthermore, CD33 expression has been demonstrated on leukemic progenitor cells, making it a promising therapeutic target for AML. Gentuzumabozogamicin is a human IgG4 CD33 monoclonal antibody conjugated to a calicheamicin derivative with a DAR of 2-3 and approximately 50% naked antibody. It was approved by the FDA in September 2017 for adults with newly diagnosed CD33-positive acute myeloid leukemia (AML), adults with relapsed or refractory AML, and pediatric patients 2 years of age and older with AML. IMGN779 (ImmunoGen, Inc., Waltham, Massachusetts) consists of a CD33 monoclonal antibody coupled to indolinobenzodiazeprine pseudodimers (alkylating agents). A clinical study (NCT02674763) has been conducted, and 50 patients have received IMGN779 with no dose-limiting toxicities (DLTs). 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, demonstrating safety and efficacy in preliminary results. BI 836858, a fully human IgG1 anti-CD33 antibody, enhances binding to FCγRIIIa to enhance its ADCC activity. Preliminary results from Phase 1b reported outcomes in 31 patients treated with BI836858, with five patients achieving a CR / complete response and four achieving morphological leukemia-free status. AMG330 (Amgen) is a CD33 / CD3 bispecific T cell engager (BiTE) antibody structure that directs cytotoxic T cells to CD33-expressing AML cells, killing target cells. It has shown efficacy 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 (GT Biopharma) (CD16 / IL-15 / CD33) enhances natural killer cell function by targeting CD33-positive malignant cells and CD33 myeloid-derived suppressor cells (MDSCs), thereby inducing an anti-tumor response targeting CD33.Currently in Phase 1 / 2 clinical study, NCT03214666, GTB-3550 (CD16 / IL-15 / CD33) Tri-Specific Killer Engager (TriKE TM AML is a highly malignant hematologic malignancy that highly expresses CD33. Currently, companies are developing monoclonal antibodies, bispecific antibodies, trispecific antibodies, and ADCs. Mylotarg has been approved by the FDA. Although, like all drug development, it presents numerous challenges, CD33 remains the most promising target for AML.

[0006] Interleukin-3 (IL-3), a major component of HGFs produced by hematologic malignant cells, binds to its cell surface receptor, IL-3R, inducing proliferation, anti-apoptosis, and differentiation signals. CD123, also known as IL-3Ra, is the α chain of IL-3R. The complex formed by IL-3Ra binding to IL-3 and recruiting the IL-3Rβ chain can cascade and activate the downstream intracellular signaling pathway, JAK2, mediating cell proliferation and survival.

[0007] CD123 is a 360-amino acid glycoprotein that also serves as the α chain of the interleukin-3 receptor (IL-3R). Together with IL-3Rβ (CD131), it forms the high-affinity IL-3R. After specifically recognizing and binding to IL-3, IL-3R promotes cell growth and proliferation, and is associated with the development of tumors, allergic inflammation, and autoimmune diseases. Studies have confirmed that CD123 is an AML-associated antigen, highly expressed in leukemia stem cells and low or absent in normal hematopoietic stem and progenitor cells, making it a potential therapeutic target for AML. Leukemia cells in AML patients can spontaneously phosphorylate and activate STAT5, promoting cell proliferation and differentiation and resisting apoptosis. Therefore, high expression of CD123 is a factor contributing to poor prognosis in AML. CSL362 is a humanized anti-CD123 monoclonal antibody and a derivative of CSL360. It uses antibody engineering technology to enhance its affinity for CD16 on the surface of natural killer (NK) cells, thereby enhancing its anti-leukemic efficacy by increasing the antibody-dependent cell-mediated cytotoxicity (ADCC) of NK cells. Animal studies have shown that CSL362 significantly inhibits the proliferation of leukemic cells in AML-transplanted mice, and CSL-362 is currently in Phase I clinical trials. In addition, there are several bispecific antibodies targeting CD123. MGD006, which simultaneously targets CD123 and CD3, has demonstrated anti-leukemic efficacy in early clinical studies and is currently being used experimentally for the treatment of refractory / relapsed AML. To date, monoclonal antibodies targeting CD123 have not been associated with significant toxic side effects in the treatment of AML. However, no anti-CD123 antibodies have been formally used in clinical treatment, and their anti-leukemic efficacy in humans remains to be determined.

[0008] Currently, drugs targeting CD123 include ADCs, bispecific antibodies, and CAR-T drugs. For example, IMGN632 is an ADC targeting CD123, which received Breakthrough Therapy Designation (BTD) from the US FDA in October 2020 for the treatment of hematological malignancies, including blastic plasmacytoid dendritic cell neoplasm (BPDCN), acute myeloid leukemia (AML), and acute lymphoblastic leukemia (ALL). Talacotuzumab is a monoclonal antibody developed by Janssen, a Johnson & Johnson subsidiary.

[0009] Bispecific antibodies (BsAbs) consist of two physically linked antigen-binding moieties that can simultaneously interact with different epitopes on the same or different antigens, exerting synergistic effects that offer advantages over monoclonal antibodies. They can also mediate a variety of specific biological effects. For example: ① Bridging immune cells and tumor cells, recruiting and activating immune cells to kill tumor cells; ② Inhibiting or stimulating multiple signaling pathways, exerting a coordinated effect; and ③ Leveraging the antibody's bivalent structure, mediating the formation of protein complexes and exerting biological effects. BsAbs do not exist naturally but are instead produced through cell fusion or recombinant DNA technology. Due to their specificity and bifunctionality, they have become a research hotspot in the field of antibody engineering, with broad application prospects in areas such as tumor therapy and autoimmune diseases. Summary of the Invention

[0010] The present invention provides an anti-CD33 single-domain antibody, an anti-CD123 antibody, a polypeptide targeting CD33 and CD123 molecules, and uses thereof. The present invention provides an anti-CD33 single-domain antibody comprising a CDR1, a CDR2, and a CDR3 selected from the following amino acid sequences:

[0011] CDR1 shown in any one of SEQ ID NOs: 9-11;

[0012] CDR2 shown in any one of SEQ ID NO: 12 and SEQ ID NO: 13;

[0013] CDR3 shown in any one of SEQ ID NOs: 14-16;

[0014] 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.

[0015] Preferably, the amino acid sequences of CDR1, CDR2 and CDR3 are selected from the following groups:

[0016] (1) CDR1 shown in SEQ ID NO: 9, CDR2 shown in SEQ ID NO: 12, and CDR3 shown in SEQ ID NO: 14;

[0017] (2) CDR1 shown in SEQ ID NO: 10, CDR2 shown in SEQ ID NO: 13, and CDR3 shown in SEQ ID NO: 15;

[0018] (3) CDR1 shown in SEQ ID NO: 11, CDR2 shown in SEQ ID NO: 13, and CDR3 shown in SEQ ID NO: 16;

[0019] (4) CDR1 shown in SEQ ID NO: 11, CDR2 shown in SEQ ID NO: 13, and CDR3 shown in SEQ ID NO: 16.

[0020] All of the above sequences can be replaced by sequences having "at least 80% homology" to the sequence or sequences with only one or a few amino acid substitutions; preferably "at least 85% homology", more preferably "at least 90% homology", more preferably "at least 95% homology", and most preferably "at least 98% homology".

[0021] Preferably, the single domain antibody consists of heavy chains.

[0022] In one embodiment, one to five amino acid residues in any one or more CDRs can be substituted with their conservative amino acids. Specifically, in CDR1, one to five amino acid residues can be substituted with their conservative amino acids; in CDR2, one to five amino acid residues can be substituted with their conservative amino acids; and in CDR3, one to five amino acid residues can be substituted with their conservative amino acids.

[0023] As used herein, the term "sequence homology" refers to the extent to which two (nucleotide or amino acid) sequences have identical residues at identical positions in an alignment, and is typically expressed as a percentage. Preferably, homology is determined over the entire length of the sequences being compared. Thus, two copies of identical sequences have 100% homology.

[0024] In some embodiments, sequences that replace 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 variations include (but are not limited to): deletion, insertion and / or substitution of one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids, and addition of one or more (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 CDR1, CDR2 and CDR3 combination in a single domain antibody, technicians can consider so-called "conservative" amino acid substitutions. 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 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 of one or a few amino acids within the following groups (a)-(d) by another or a few amino acids within 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 or Gln; Phe is replaced by Met, Leu, or Tyr; Trp is replaced by Tyr; Tyr is replaced by Phe or 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 will appreciate that the framework region sequences FR1-4 are not immutable, and the sequences of FR1-4 may be conservative sequence variants of the sequences disclosed herein.

[0025] The meaning of "anti-CD33 single-domain antibodies" in the present invention includes not only intact 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 refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides 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) polypeptides having a substituent group in one or more amino acid residues, or (iii) polypeptides formed by fusion of a mature polypeptide with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein formed with an Fc tag). Based on the teachings of this document, these fragments, derivatives, and analogs are well known to those skilled in the art.

[0026] 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:

[0027] FR1 shown in any one of SEQ ID NOs: 17-18;

[0028] FR2 shown in any one of SEQ ID NOs: 19-21;

[0029] FR3 shown in any one of SEQ ID NOs: 22-24;

[0030] FR4 shown in SEQ ID NO:25-26;

[0031] 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.

[0032] Another aspect of the present invention provides an amino acid sequence of a single-domain antibody capable of binding to CD33, wherein the amino acid sequence of the single-domain antibody is selected from any one of SEQ ID NOs: 1-4, or has at least 80% sequence homology with the amino acid sequence shown in SEQ ID NOs: 1-4, and is capable of specifically binding to the CD33 protein.

[0033] In one embodiment, the anti-CD33 single domain antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to a sequence selected from SEQ ID NOs: 1-4 or to an amino acid sequence of SEQ ID NOs: 1-4, and is capable of specifically binding to a CD33 protein.

[0034] In one embodiment, the anti-CD33 single domain antibody is an Fc fusion antibody or a humanized antibody.

[0035] Another aspect of the present invention provides a recombinant protein comprising any one of the aforementioned antibodies.

[0036] In one embodiment, the recombinant protein comprises the aforementioned anti-CD33 single domain antibody.

[0037] Preferably, the recombinant protein may be a single-domain antibody as shown in the aforementioned SEQ ID NOs: 1-4, or a single-domain antibody having at least 80% homology with SEQ ID NOs: 1-4, 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 NOs: 1-4; a multivalent antibody may be composed of one of the sequences in SEQ ID NOs: 1-4 repeated several times; multispecific antibodies include but are not limited to bispecific antibodies, and trispecific antibodies; in addition, the recombinant protein may be a fragment, derivative, or analog of the aforementioned antibodies.

[0038] Another aspect of the present invention provides an isolated nucleic acid molecule encoding any one of the aforementioned antibodies or recombinant proteins.

[0039] Preferably, the nucleotide sequence thereof is as shown in SEQ ID NO: 5-8, respectively, or has at least 95% sequence homology with SEQ ID NO: 5-8.

[0040] In one embodiment, the nucleic acid molecule encoding the anti-CD33 single domain antibody is selected from SEQ ID NOs: 5-8, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to a nucleotide sequence selected from SEQ ID NOs: 5-8, and the encoded anti-CD33 single domain antibody is capable of specifically binding to an anti-CD33 protein.

[0041] Another aspect of the present invention provides an expression vector comprising a nucleotide molecule encoding any one of the aforementioned antibodies, or the provided nucleotide molecule.

[0042] Preferably, the expression vector comprises a nucleotide molecule encoding an anti-CD33 single domain antibody, Fc fusion antibody or humanized antibody, and the nucleotide sequence is selected from SEQ ID NOs: 5-8 or has at least 80% sequence homology with any one of SEQ ID NOs: 5-8.

[0043] In a preferred embodiment, the expression vector used is RJK-V4-hFC1 (a nucleotide molecule encoding an anti-CD33 single-domain antibody or an Fc fusion antibody or a humanized antibody is integrated into RJK-V4-hFC1 by genetic engineering). Other general expression vectors can also be selected as needed.

[0044] Another aspect of the present invention provides a host cell, which expresses any one of the aforementioned antibodies, or contains the provided nucleotide molecule or the provided expression vector.

[0045] Preferably, the host cell is capable of expressing the aforementioned anti-CD33 single domain antibody, Fc fusion antibody or humanized antibody, or contains the aforementioned expression vector.

[0046] In another preferred embodiment, the host cell is a bacterial cell, a fungal cell or a mammalian cell.

[0047] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell, including bacteria and fungi.

[0048] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, mammalian cells, bacteriophages, or a combination thereof.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] In another preferred embodiment, the host cells are suspension ExpiCHO-S cells.

[0053] In another preferred embodiment, the host cells are suspension 293F cells.

[0054] Another aspect of the present invention provides a pharmaceutical composition comprising any of the aforementioned antibodies, the provided recombinant protein, the provided nucleic acid, the provided expression vector, the provided host cell, or a combination thereof; and a pharmaceutically acceptable carrier.

[0055] Preferably, the pharmaceutical composition comprises the aforementioned anti-CD33 binding single domain antibody and a pharmaceutically acceptable carrier. Typically, these substances can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally determined by the isoelectric point of the antibody (the pH of the aqueous carrier medium should deviate from the isoelectric point of the antibody by approximately 2).

[0056] The pharmaceutical composition of the present invention can be directly used to bind to anti-CD33 protein molecules.

[0057] The pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the aforementioned single domain antibody 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 be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions.

[0058] Another aspect of the present invention provides a medicament for treating a disease, comprising the provided pharmaceutical composition.

[0059] Preferably, the aforementioned single-domain antibody for binding to CD33 protein is contained as an active ingredient.

[0060] Another aspect of the present invention provides a kit for detecting CD33 levels, comprising any one of the aforementioned anti-CD33 single-domain antibodies.

[0061] In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, a buffer, and the like.

[0062] In a preferred embodiment, the kit includes an antibody that recognizes the CD33 protein, a lysis medium for dissolving the sample, and general 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.

[0063] In a preferred embodiment, the kit further contains a secondary antibody and an enzyme or fluorescent or radioactive label for detection, and a buffer.

[0064] In a preferred embodiment, the second antibody of the kit can be an antibody (as an anti-antibody) of the aforementioned anti-CD33 single domain antibody, and can be a single domain antibody, a monoclonal antibody, a polyclonal antibody or any other form of antibody.

[0065] In another aspect, the present invention provides a method for producing an anti-CD33 single domain antibody, comprising the steps of:

[0066] (a) culturing the host cell 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

[0067] (b) isolating or recovering the anti-CD33 single domain antibody from the culture; and

[0068] (c) optionally, purifying and / or modifying the anti-CD33 single domain antibody obtained in step (b).

[0069] Another aspect of the present invention provides use of the aforementioned anti-CD33 single-domain antibody or the aforementioned pharmaceutical composition in the preparation of a medicament for treating tumor diseases.

[0070] In a preferred embodiment, the disease is a hematological tumor.

[0071] In a preferred embodiment, the disease is acute myeloid leukemia.

[0072] Another aspect of the present invention provides a method for treating a disease using the aforementioned anti-CD33 single-domain antibody or the aforementioned pharmaceutical composition.

[0073] In a preferred embodiment, the disease is a hematological tumor.

[0074] In a preferred embodiment, the disease is acute myeloid leukemia.

[0075] The purpose of the present invention is to provide a single-domain antibody that can specifically bind to CD123 and its use.

[0076] Another aspect of the present invention provides an anti-CD123 single-domain antibody comprising CDR1, CDR2, and CDR3 selected from the following amino acid sequences:

[0077] CDR1 shown in any one of SEQ ID NOs:41-46;

[0078] CDR2 shown in any one of SEQ ID NOs:47-51;

[0079] CDR3 shown in any one of SEQ ID NOs: 52-57;

[0080] 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 CD123 binding affinity.

[0081] Preferably, the amino acid sequences of CDR1, CDR2 and CDR3 are selected from the following groups:

[0082] (1) CDR1 shown in SEQ ID NO:41, CDR2 shown in SEQ ID NO:47, and CDR3 shown in SEQ ID NO:52;

[0083] (2) CDR1 shown in SEQ ID NO:42, CDR2 shown in SEQ ID NO:47, and CDR3 shown in SEQ ID NO:52;

[0084] (3) CDR1 shown in SEQ ID NO:43, CDR2 shown in SEQ ID NO:48, and CDR3 shown in SEQ ID NO:53;

[0085] (4) CDR1 shown in SEQ ID NO:44, CDR2 shown in SEQ ID NO:49, and CDR3 shown in SEQ ID NO:54;

[0086] (5) CDR1 shown in SEQ ID NO:45, CDR2 shown in SEQ ID NO:50, and CDR3 shown in SEQ ID NO:55;

[0087] (6) CDR1 shown in SEQ ID NO:46, CDR2 shown in SEQ ID NO:51, and CDR3 shown in SEQ ID NO:56;

[0088] (7) CDR1 shown in SEQ ID NO:46, CDR2 shown in SEQ ID NO:51, and CDR3 shown in SEQ ID NO:57.

[0089] Preferably, the single domain antibody consists of heavy chains.

[0090] All of the above sequences can be replaced by sequences having at least 80% homology with the sequences or sequences with only one or a few amino acid substitutions; preferably at least 85% homology, more preferably at least 90% homology, more preferably at least 95% homology, and most preferably at least 98% homology.

[0091] In one embodiment, one to five amino acid residues in any one or more CDRs can be substituted with their conservative amino acids. Specifically, in CDR1, one to five amino acid residues can be substituted with their conservative amino acids; in CDR2, one to five amino acid residues can be substituted with their conservative amino acids; and in CDR3, one to five amino acid residues can be substituted with their conservative amino acids.

[0092] As used herein, the term "sequence homology" refers to the extent to which two (nucleotide or amino acid) sequences have identical residues at identical positions in an alignment, and is typically expressed as a percentage. Preferably, homology is determined over the entire length of the sequences being compared. Thus, two copies of identical sequences have 100% homology.

[0093] In some embodiments, sequences that replace 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 variations include (but are not limited to): deletion, insertion and / or substitution of one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids, and addition of one or more (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 CDR1, CDR2 and CDR3 combination in a single domain antibody, technicians can consider so-called "conservative" amino acid substitutions. 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 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 of one or a few amino acids within the following groups (a)-(d) by another or a few amino acids within 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 or Gln; Phe is replaced by Met, Leu, or Tyr; Trp is replaced by Tyr; Tyr is replaced by Phe or 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 will appreciate that the framework region sequences FR1-4 are not immutable, and the sequences of FR1-4 may be conservative sequence variants of the sequences disclosed herein.

[0094] The meaning of “single domain antibody against CD123” in the present invention includes not only complete single domain antibodies, but also fragments, derivatives and analogs of single domain antibodies against CD123. 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 can be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by a genetic code, or (ii) a polypeptide having a substituent 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 extends 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 for purifying the polypeptide, or a fusion protein formed with an Fc tag). According to the teachings herein, these fragments, derivatives and analogs belong to the scope known to those skilled in the art.

[0095] 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:

[0096] FR1 or a variant of FR1 shown in any one of SEQ ID NOs: 17-18, 58;

[0097] FR2 or a variant of FR2 shown in any one of SEQ ID NOs: 59-64;

[0098] FR3 or a variant of FR3 shown in any one of SEQ ID NOs: 65-71;

[0099] FR4 or a variant of FR4 shown in any one of SEQ ID NOs: 25-26.

[0100] 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 CD123 binding affinity.

[0101] On the other hand, the present invention provides an amino acid sequence of a single-domain antibody that can bind to CD123, wherein the amino acid sequence of the single-domain antibody is as shown in SEQ ID NO: 27-33, or has at least 80% sequence homology with the amino acid sequence of SEQ ID NO: 27-33, and can specifically bind to the CD123 protein.

[0102] In one embodiment, the anti-CD123 single domain antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology to an amino acid sequence selected from SEQ ID NOs: 27-33 or SEQ ID NOs: 27-33, and is capable of specifically binding to a CD123 protein.

[0103] In one embodiment, the anti-CD123 single domain antibody is a humanized antibody or an Fc fusion antibody.

[0104] Another aspect of the present invention provides a recombinant protein comprising any one of the aforementioned antibodies.

[0105] In one embodiment, the recombinant protein comprises the aforementioned anti-CD123 single domain antibody.

[0106] Preferably, the recombinant protein can be a single-domain antibody as shown in the aforementioned SEQ ID NOs: 27-33, or a single-domain antibody having at least 80% homology with SEQ ID NOs: 27-33, or a multi-epitope antibody, a multi-specific antibody, and a multivalent antibody; for example, a multi-epitope antibody can be composed of more than one sequence in SEQ ID NOs: 1-7; a multivalent antibody can be composed of one of the sequences in SEQ ID NOs: 27-33 repeated several times; multispecific antibodies include but are not limited to bispecific antibodies, and trispecific antibodies; in addition, the recombinant protein can be a fragment, derivative, and analog of the aforementioned antibodies.

[0107] Another aspect of the present invention provides an isolated nucleic acid molecule encoding any of the aforementioned antibodies or recombinant proteins.

[0108] Preferably, the nucleic acid molecule sequence is as shown in SEQ ID NO: 8-14, or has at least 95% sequence homology with SEQ ID NO: 8-14.

[0109] In one embodiment, the nucleic acid molecule encoding an anti-CD123 single-domain antibody 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 to a nucleotide sequence selected from SEQ ID NOs: 8-14, and the anti-CD123 single-domain antibody encoded thereby can specifically bind to an anti-CD123 protein.

[0110] Another aspect of the present invention provides an expression vector comprising a nucleic acid molecule encoding any of the aforementioned antibodies, or the provided nucleic acid molecule.

[0111] Preferably, the expression vector comprises a nucleotide molecule encoding an anti-CD123 single domain antibody or Fc fusion antibody or humanized antibody, and the nucleotide sequence is selected from SEQ ID NO: 34-40, or has at least 80% sequence homology with any one of SEQ ID NO: 34-40.

[0112] In a preferred embodiment, the expression vector used is RJK-V4-hFC1 (a nucleotide molecule encoding an anti-CD123 single-domain antibody or its Fc fusion antibody or humanized antibody is integrated into RJK-V4-hFC1 by genetic engineering means). Other general expression vectors can also be selected as needed.

[0113] Another aspect of the present invention provides a host cell, which expresses any of the aforementioned antibodies, or contains the provided nucleic acid molecule or the provided expression vector.

[0114] Preferably, the host cell is capable of expressing the aforementioned anti-CD123 single domain antibody, Fc fusion antibody or humanized antibody, or contains the aforementioned expression vector.

[0115] In another preferred embodiment, the host cell is a bacterial cell, a fungal cell or a mammalian cell.

[0116] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell, including bacteria and fungi.

[0117] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, mammalian cells, bacteriophages, or a combination thereof.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] In another preferred embodiment, the host cells are suspension ExpiCHO-S cells.

[0122] In another preferred embodiment, the host cells are suspension 293F cells.

[0123] Another aspect of the present invention provides a pharmaceutical composition comprising any of the aforementioned antibodies, the provided recombinant protein, the provided nucleic acid, the provided expression vector, the provided host cell, or a combination thereof; and a pharmaceutically acceptable carrier.

[0124] Preferably, the pharmaceutical composition comprises the aforementioned single domain antibody that binds to anti-CD123 and a pharmaceutically acceptable carrier. Typically, 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).

[0125] The pharmaceutical composition of the present invention can be directly used to bind to anti-CD123 protein molecules.

[0126] The pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the aforementioned single domain antibody 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 be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions.

[0127] Another aspect of the present invention provides a medicament for treating a disease, comprising the provided pharmaceutical composition.

[0128] Preferably, the aforementioned single domain antibody for binding to CD123 protein is contained as an active ingredient.

[0129] Another aspect of the present invention provides a kit for detecting CD123 levels, comprising any one of the aforementioned anti-CD123 single-domain antibodies.

[0130] In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, a buffer, and the like.

[0131] In a preferred embodiment, the kit includes an antibody that recognizes the CD123 protein, a lysis medium for dissolving the sample, and general 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.

[0132] In a preferred embodiment, the kit further contains a secondary antibody and an enzyme or fluorescent or radioactive label for detection, and a buffer.

[0133] In a preferred embodiment, the second antibody of the kit can be the aforementioned anti-CD123 single domain antibody (as an anti-antibody), which can be a single domain antibody, a monoclonal antibody, a polyclonal antibody or any other form of antibody.

[0134] In another aspect, the present invention provides a method for producing an anti-CD123 single domain antibody, comprising the steps of:

[0135] (a) culturing the host cell of the present invention under conditions suitable for producing the single domain antibody, thereby obtaining a culture containing the anti-CD123 single domain antibody; and

[0136] (b) isolating or recovering the anti-CD123 single domain antibody from the culture; and

[0137] (c) optionally, purifying and / or modifying the anti-CD123 single domain antibody obtained in step (b).

[0138] Another aspect of the present invention provides use of the aforementioned anti-CD123 single-domain antibody or the aforementioned pharmaceutical composition in the preparation of a medicament for treating a disease.

[0139] In a preferred embodiment, the disease is a hematological oncological disease.

[0140] In a preferred embodiment, the disease is acute myeloid leukemia.

[0141] Another aspect of the present invention provides a method for treating a disease using the aforementioned anti-CD123 single-domain antibody or the aforementioned pharmaceutical composition.

[0142] In a preferred embodiment, the disease is a hematological oncological disease.

[0143] In a preferred embodiment, the disease is acute myeloid leukemia.

[0144] The present invention provides a polypeptide targeting CD123 and CD33, the polypeptide comprising a first binding portion that specifically binds to CD33 and a second binding portion that specifically binds to CD123, wherein the polypeptide comprises or consists of at least two immunoglobulin single variable domains, the variable domains comprising three complementarity determining regions CDR1, CDR2 and CDR3, and the complementarity determining regions comprising the following amino acid sequence:

[0145] The CDR1 of the first binding moiety comprises the amino acid sequence of SEQ ID NO: 11, the CDR2 comprises the amino acid sequence of SEQ ID NO: 13, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 16;

[0146] the CDR1 of the second binding moiety comprises the amino acid sequence of SEQ ID NO:46, the CDR2 comprises the amino acid sequence of SEQ ID NO:51, and the CDR3 comprises the amino acid sequence of SEQ ID NO:57;

[0147] The amino acid sequence includes derivative sequences formed by optionally adding, deleting, modifying and / or replacing 1-3 amino acids, and capable of retaining the binding affinity to CD123 and CD33, respectively.

[0148] In some embodiments, the variable domain is the variable domain of a heavy chain antibody, wherein the variable domain of the first binding portion comprises the amino acid sequence of SEQ ID NO:4 or is at least 90% identical to the amino acid sequence of SEQ ID NO:4; and the variable domain of the second binding portion comprises the amino acid sequence of SEQ ID NO:33 or is at least 90% identical to the amino acid sequence of SEQ ID NO:33.

[0149] In some implementations, wherein:

[0150] a) the variable domain of the first binding moiety consists of the amino acid sequence of SEQ ID NO: 4;

[0151] b) the variable domain of the second binding moiety consists of the amino acid sequence of SEQ ID NO: 33.

[0152] The present invention provides an anti-CD33 single-domain antibody, which consists of the amino acid sequence of SEQ ID NO: 1.

[0153] The present invention provides an anti-CD123 single-domain antibody, which consists of the amino acid sequence of SEQ ID NO: 2.

[0154] Single-domain antibodies (sdAbs) are derived from naturally occurring single-domain antigen-binding molecules, which are referred to as heavy chain antibodies lacking light chains (also referred to herein as "heavy chain antibodies only"). Single-domain antibodies are antibodies that naturally lack light chains in the peripheral blood of camels. These antibodies only contain a heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, but they are not as easy to stick to each other as artificially modified single-chain antibody fragments (scFvs), and even aggregate into clumps. For the sake of clarity, the variable domains derived from heavy chain molecules that naturally lack light chains are referred to herein as VHHs to distinguish them from conventional VHs of four-chain immunoglobulins. Such VHH molecules can be derived from antibodies produced in camel species, for example, camels, llamas, llamas, dromedaries, alpacas and guanacos, as well as other species other than camelids that can produce heavy chain molecules that naturally lack light chains. VHHs produced by the above species are all within the scope of this application. In other embodiments, the VHH may be derived from a condricthoid, and in other embodiments, the VHH may be derived from a shark. A single domain antibody may also comprise only a heavy chain variable region (VHH) without CH2 and CH3 regions.

[0155] In some embodiments, single domain antibodies (sdAbs) are recombinant, CDR-grafted, humanized, camelized, deimmunized, and / or generated in vitro (e.g., by phage display selection). In some embodiments, sdAbs are human sdAbs produced by transgenic mice or rats expressing human heavy chain fragments.

[0156] In some embodiments, the VHH comprises a naturally occurring sdAb or its VHH fragment or derivative thereof, such as a camel sdAb or its VHH fragment, or a humanized sdAb or its VHH fragment derived from a camel sdAb. In one embodiment, the sdAb is derived from a llama. In one embodiment, the sdAb is further engineered to remove sequences not typically present in human antibodies (such as CDR regions or CDR-FR junctions).

[0157] On the other hand, the present invention provides a bispecific antibody, which comprises a first full-length antibody and a second full-length antibody, wherein the first full-length antibody comprises a heavy chain variable domain and an Fc region polypeptide that specifically binds to a first binding portion of CD33, and the second full-length antibody comprises a heavy chain variable domain and an Fc region polypeptide that specifically binds to a second binding portion of CD123, wherein the heavy chain variable domain comprises three complementarity determining regions (CDR1, CDR2, and CDR3), and the CDR1 of the first full-length antibody comprises the amino acid sequence of SEQ ID NO: 11, the CDR2 comprises the amino acid sequence of SEQ ID NO: 13, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 16; the CDR1 of the second full-length antibody comprises the amino acid sequence of SEQ ID NO: 46, the CDR2 comprises the amino acid sequence of SEQ ID NO: 51, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 57.

[0158] The amino acid sequence includes derivative sequences formed by optionally adding, deleting, modifying and / or replacing 1-3 amino acids, and capable of retaining the binding affinity to CD123 and CD33, respectively.

[0159] The FcγIIIaR binding of an antibody or Fc-region fusion polypeptide can be modified by altering amino acid residues at non-corresponding positions in the respective Fc-region polypeptides, as these modifications work together in FcγIIIaR binding. The present invention has discovered that by preparing two human IgG Fc-region polypeptides and modifying (mutating / substituting) the amino acid residues of each Fc-region polypeptide, variant Fc-region polypeptides can be generated. Furthermore, by pairing the two variant Fc-region polypeptides, the formation of a structurally stable heterodimer is facilitated.

[0160] The present invention found that when the two variant Fc region polypeptides are fused with different VHHs, they can form stable heterodimers; at the same time, based on the fact that both variant Fc regions are derived from the Fc region of human IgG, both variant Fc region polypeptides have Fc-mediated related functions. The above-mentioned heterodimers are prepared into antibodies (bispecific antibodies, etc.), which present an asymmetric structure. Each chain in the antibody can play a role against different antigens, and each chain has Fc-mediated functions. Therefore, the antibody with an asymmetric structure reported in this article not only has a high degree of structural stability, but also has functional advantages over symmetrical bispecific antibodies or multispecific antibodies (two polypeptides are completely symmetrical). The targeting function of the antibody and the Fc-mediated function are not affected by each other. As reported herein, it is effective to use antibodies and Fc region fusion polypeptides with different antigen binding regions to treat diseases such as cancer.

[0161] In some embodiments, the amino acid difference between the first variant Fc region polypeptide and the second variant Fc region polypeptide occurs in the CH3 domain of a human IgG class Fc region.

[0162] The human IgG class Fc region includes a portion of the hinge region, the CH2 region, and the CH3 region. In some embodiments, a) the first variant Fc region polypeptide has amino acid differences relative to the human IgG class Fc region polypeptide in the CH3 domain of the human IgG class Fc region;

[0163] and b) the second variant Fc region polypeptide has amino acid differences relative to the human IgG class Fc region polypeptide present in the CH3 domain of the human IgG class Fc region.

[0164] In some embodiments, the Fc region polypeptide is derived from a human IgG class Fc region polypeptide, which Fc region polypeptide is derived from a human IgG class Fc region polypeptide, comprising a modified first variant Fc region polypeptide and a modified second variant Fc region polypeptide; wherein the carboxyl terminus of the heavy chain variable domain of the first binding moiety is coupled to the amino terminus of the modified first variant Fc region polypeptide, and the carboxyl terminus of the heavy chain variable domain of the second binding moiety is coupled to the amino terminus of the modified second variant Fc region polypeptide.

[0165] In some embodiments: a) the variable domain of the first binding moiety consists of the amino acid sequence of SEQ ID NO:4; b) the variable domain of the second binding moiety consists of the amino acid sequence of SEQ ID NO:33.

[0166] In some embodiments, the antibody comprises a first full-length antibody consisting of the amino acid sequence of SEQ ID NO:74 and a second full-length antibody consisting of the amino acid sequence of SEQ ID NO:75.

[0167] Another aspect of the present invention provides nucleotide sequences encoding a first full-length antibody and a second full-length antibody. In some embodiments, the nucleotide sequence encoding the first full-length antibody is shown in SEQ ID NO: 76, and the nucleotide sequence encoding the second full-length antibody is shown in SEQ ID NO: 77.

[0168] In some embodiments, the present invention provides a nucleotide molecule encoding any of the aforementioned polypeptides or the aforementioned antibodies.

[0169] Another aspect of the present invention provides a vector comprising a nucleotide molecule encoding any one of the aforementioned polypeptides or the aforementioned antibody, or the aforementioned nucleotide molecule.

[0170] Another aspect of the present invention provides a host cell, which can express the aforementioned polypeptide or the aforementioned antibody, or the aforementioned vector, or contains the aforementioned nucleic acid, or contains the aforementioned expression vector.

[0171] In some embodiments, the host cell comprises a prokaryotic cell, a eukaryotic cell, or a bacteriophage.

[0172] In some embodiments, the host cell is selected from the group consisting of E. coli, yeast cells, mammalian cells, bacteriophage, or a combination thereof.

[0173] In some embodiments, the prokaryotic cell is selected from the group consisting of Escherichia coli, Bacillus subtilis, Lactobacillus, Streptomyces, Proteus mirabilis, or a combination thereof.

[0174] In some embodiments, the eukaryotic cell is selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, or a combination thereof.

[0175] In some embodiments, 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.

[0176] In some embodiments, the host cells are suspension ExpiCHO-S cells.

[0177] In some embodiments, the host cells are suspension 293F cells.

[0178] Another aspect of the present invention provides a pharmaceutical composition comprising any of the aforementioned polypeptides, any of the aforementioned antibodies, any of the aforementioned nucleic acids, any of the aforementioned expression vectors, any of the aforementioned host cells, or a combination thereof; and a pharmaceutically acceptable carrier.

[0179] Another aspect of the present invention provides use of any of the aforementioned polypeptides, any of the aforementioned antibodies, or the aforementioned pharmaceutical compositions in the preparation of a medicament for treating a disease.

[0180] In some embodiments, the disease is a tumor.

[0181] In some embodiments, the disease is a hematological neoplasm.

[0182] In some embodiments, the disease is acute myeloid leukemia, human multiple myeloma, human monocytic leukemia, human acute lymphoblastic leukemia, adult B-type acute lymphoblastic leukemia, Ph+ acute lymphoblastic leukemia cell line, and Epstein-Barr virus-infected human peripheral lymphocyte cell line.

[0183] Another aspect of the present invention provides a method for producing an antibody, comprising: (a) culturing the aforementioned host cell and (b) recovering the aforementioned antibody.

[0184] Another aspect of the present invention provides an immunoconjugate comprising any of the aforementioned polypeptides or any of the aforementioned bispecific antibodies, or a combination thereof.

[0185] In some embodiments, the immunoconjugate includes a cytotoxin, a biologically active protein, or a radioisotope.

[0186] Another aspect of the present invention provides a method for treating tumors using any of the aforementioned polypeptides, any of the aforementioned bispecific antibodies, or any of the aforementioned pharmaceutical compositions.

[0187] Another aspect of the present invention provides an antibody-drug conjugate comprising any of the aforementioned polypeptides or any of the aforementioned antibodies, or a combination thereof.

[0188] In some embodiments, the present invention provides a method for treating tumors using the aforementioned polypeptide or the aforementioned antibody.

[0189] In some embodiments, the tumor is a blood tumor, and further, the blood tumor is acute myeloid leukemia, human multiple myeloma, human monocytic leukemia, human acute lymphoblastic leukemia, adult type B acute lymphoblastic leukemia, Ph+ acute lymphoblastic leukemia cell line and Epstein-Barr virus-infected human peripheral lymphocyte line. Beneficial effects

[0190] Compared with the prior art, the present invention has the following beneficial effects:

[0191] (1) The single-domain antibody of the present invention is specific for the anti-CD33 protein with a correct spatial structure.

[0192] (2) The single-domain antibody obtained by the present invention has a flexible expression system selection and can be expressed in either 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 subsequent production cost.

[0193] (3) The single-domain antibodies obtained by the present invention are simple 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.

[0194] (4) The single-domain antibody of the present invention is specific for the anti-CD123 protein with the correct spatial structure.

[0195] (5) The single-domain antibody obtained by the present invention has a flexible expression system selection and can be expressed in either 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 subsequent production cost.

[0196] (6) The single-domain antibodies obtained in the present invention have a wider affinity range. Before affinity maturation, their affinity range can range from nM level to pM level, providing multiple options for antibodies with different uses in the future.

[0197] (7) The anti-CD123 / anti-CD33 bispecific polypeptide prepared by the present invention can target CD123 protein and CD33 protein respectively. When used as an anti-tumor drug, it showed significant tumor inhibitory effect in the study of tumor growth inhibition rate in the KG-1 human leukemia cell xenograft tumor model, and still showed significant inhibitory effect after drug withdrawal. It also showed significant tumor inhibitory effect in the in vivo efficacy study of the human acute myeloid leukemia MV4-11 cell subcutaneous xenograft tumor CB17 SCID mouse model.

[0198] (8) The anti-CD123 / anti-CD33 bispecific polypeptide prepared by the present invention forms an asymmetric bispecific antibody by fusing the Fc region and has multiple functions: 1) Each chain has independent targeting: it can target CD123 and CD33 proteins respectively; 2) Each chain has ADCC effect, and the design based on the asymmetric bispecific antibody structure can ensure that the targeting function of the antibody and the Fc-mediated ADCC effector function do not affect each other.

[0199] (9) The anti-CD123 / anti-CD33 bispecific polypeptide prepared by the present invention shows good ADCC effect on blood tumor cell lines such as KG-1 (human acute myeloid leukemia cell line), RPMI-8226 (human multiple myeloma cell line), MV-4-11 (human acute myeloid leukemia cell line), THP-1 (human monocytic leukemia cell line), HAL01 (human acute lymphoblastic leukemia cell line), NALM-6 (adult B-type acute lymphoblastic leukemia cell line), SUP-B15 (Ph+ acute lymphoblastic leukemia cell line) and JVM-2 (Epstein-Barr virus-infected human peripheral lymphocyte cell line). BRIEF DESCRIPTION OF THE DRAWINGS

[0200] In order to more clearly illustrate the technical solution of the present application, the following is a brief introduction to the drawings required for use in the embodiments. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0201] Figure 1 shows the enrichment of the library for CD33-targeted antibody screening; P / N = the number of monoclonal bacteria grown after phage eluted from the positive wells in biopanning infected with TG1 bacteria / the number of monoclonal bacteria grown after phage eluted from the negative wells infected with TG1 bacteria. This parameter gradually increases after enrichment occurs; I / E = the total amount of phage added to the positive wells in each round of biopanning / the total amount of phage eluted from the positive wells in each round of biopanning. This parameter gradually approaches 1 after enrichment occurs.

[0202] FIG2 is a graph showing the dose-effect curve of antigen binding of 1B1 and 1B10 antibodies;

[0203] FIG3 is a graph showing the dose-effect curve of Tab1 and Tab3 antibody antigen binding;

[0204] FIG4 is a graph showing a hIgG antibody antigen binding dose-effect curve;

[0205] FIG5 is a graph showing the ADCC effect of the 1B1 antibody in Example 1;

[0206] FIG6 is a graph showing the ADCC effect of the 1B10 antibody in the embodiment;

[0207] FIG7 is a graph showing the ADCC effect of the 1B10V4 antibody in the example.

[0208] FIG8 is a graph showing the ADCC effect of the 1B10V4-F57 antibody in the embodiment;

[0209] FIG9 is a graph showing the ADCC effect of the Tab3 antibody in the examples.

[0210] Figure 10 shows the enrichment of the library for screening of antibodies targeting CD123 in the embodiment; wherein P / N = the number of monoclonal bacteria grown after phage eluted from the positive wells in bio-panning infected with TG1 bacteria / the number of monoclonal bacteria grown after phage eluted from the negative wells infected with TG1 bacteria, this parameter will gradually increase after enrichment occurs; I / E = the total amount of phage added to the positive wells in each round of bio-panning / the total amount of phage eluted from the positive wells in each round of bio-panning, this parameter will gradually approach 1 after enrichment occurs;

[0211] FIG11 is a graph showing the antibody-antigen binding dose-effect curves of Tab1, Tab4, hIgG, 1A2, and 1A5;

[0212] FIG12 is a graph showing the antibody-antigen binding dose-effect curve of 1A8;

[0213] FIG13 is a graph showing the antibody-antigen binding dose-effect curve of 1B5;

[0214] FIG14 is a graph showing the antibody-antigen binding dose-effect curves of 2B1 and 2B6;

[0215] FIG15 is a graph showing the ADCC effect determination of Tab1, Tab2, Tab3, Tab4, hIgG, and 1A2 in the Examples;

[0216] FIG16 is a graph showing the ADCC effect determination of 1A5, 1A8, 1B5, 2B1, and 2B6 in the Examples;

[0217] FIG17 is a graph showing the ADCC effect of 2B6-V162 in the Examples;

[0218] FIG18 is a graph showing the ADCC effect measurement of Tab4 and hIgG in the Examples.

[0219] Figure 19 shows the Fc amino acid numbering based on the Kabat numbering system, where the wild-type human Fc region is selected from IgG1;

[0220] FIG20 is a schematic structural diagram of the anti-CD33 and CD123 bispecific antibody provided by the present application;

[0221] Figure 21 is the ADCC effect curve of the tested anti-CD123 / anti-CD33 bispecific antibody molecule (tumor cell KG-1);

[0222] Figure 22 is the ADCC effect curve of the tested anti-CD123 / anti-CD33 bispecific antibody molecule (tumor cells RPMI-8226);

[0223] Figure 23 is the ADCC effect curve of the tested anti-CD123 / anti-CD33 bispecific antibody molecule (tumor cells MV-4-11);

[0224] FIG24 is an ADCC effect curve of the tested anti-CD123 / anti-CD33 bispecific antibody molecule (tumor cell THP-1);

[0225] Figure 25 is the ADCC effect curve of the tested anti-CD123 / anti-CD33 bispecific antibody molecule (tumor cell HAL-01);

[0226] Figure 26 is the ADCC effect curve of the tested anti-CD123 / anti-CD33 bispecific antibody molecule (tumor cell NALM-6);

[0227] FIG27 is an ADCC effect curve of the tested anti-CD123 / anti-CD33 bispecific antibody molecule (tumor cell SUP-B15);

[0228] FIG28 is an ADCC effect curve of the tested anti-CD123 / anti-CD33 bispecific antibody molecule (tumor cell JVM-2). DETAILED DESCRIPTION

[0229] The present invention is further described in detail below with reference to the embodiments so that those skilled in the art can implement the invention with reference to the description.

[0230] As used herein, "single-domain antibodies" (sdAbs, also referred to as nanobodies or VHHs by 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 that have only heavy chains (which naturally do not contain light chains), single-domain antibodies derived from conventional antibodies, and engineered antibodies.

[0231] 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 species of the Camelidae family (e.g., camels, dromedaries, llamas and guanacos). Like complete antibodies, single domain antibodies are capable of selectively binding to specific antigens. Single domain antibodies can contain only the variable domain of an immunoglobulin chain, which has CDR1, CDR2 and CDR3 and a framework region.

[0232] As used herein, the term "Fc fusion antibody" refers to a novel protein produced by fusing the Fc segment of an antibody of interest with a functional protein molecule having biological activity using genetic engineering technology.

[0233] 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 by transplanting the complementarity determining regions (CDR1-3) of a target antibody into the variable region of a human antibody, or by subjecting the target antibody to amino acid mutations based on the characteristics of the human antibody framework region (FR1-4). Humanized antibodies can be produced synthetically or by site-directed mutagenesis.

[0234] In the present invention, sequences with high sequence homology to the CDR1-3 sequences disclosed herein can also be used to generate single-domain antibodies against CD33. In some embodiments, sequences having "at least 80% homology," or "at least 85% homology," "at least 90% homology," "at least 95% homology," or "at least 98% homology" to the sequences in SEQ ID NOs: 1-4 can also achieve the objectives of the invention.

[0235] In some embodiments, sequences that replace only one or a few amino acids compared to the sequences in SEQ ID NOs: 1-4, 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 skilled person 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 of one or a few amino acids within the following groups (a)-(d) by another or a few amino acids within 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 or Gln; Phe is replaced by Met, Leu, or Tyr; Trp is replaced by Tyr; Tyr is replaced by Phe or 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.

[0236] 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.

[0237] Immunoglobulin single variable domain

[0238] The term "immunoglobulin single variable domain" is used interchangeably with "single variable domain" to define an immunoglobulin molecule in which the antigen binding site is present on and formed by a single immunoglobulin domain. This distinguishes a "single variable domain" from a "conventional" immunoglobulin (e.g., monoclonal antibody) or a fragment thereof (such as Fab, Fab', F(ab')2, scFv, di-scFv), in which two immunoglobulin domains, in particular two variable domains, interact to form an antigen binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (VH) and the light chain variable domain (VL) interact to form an antigen binding site. In this case, the complementarity determining regions (CDRs) of both VH and VL will contribute to the antigen binding site, i.e., a total of 6 CDRs will participate in the formation of the antigen binding site.

[0239] In view of the above definition, the antigen-binding domain of a conventional 4-chain antibody (such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art) or the antigen-binding domain of a Fab fragment, F(ab')2 fragment, Fv fragment (such as a disulfide-linked Fv or scFv fragment) or diabody (all known in the art) derived from such a conventional 4-chain antibody would not generally be considered to be a "single variable domain" because, in these cases, binding to the corresponding epitope of the antigen generally does not occur through one single immunoglobulin domain, but rather through a pair of associated immunoglobulin domains, such as a light and heavy chain variable domain, i.e., through a VH-VL pair of immunoglobulin domains that bind together to an epitope of the corresponding antigen.

[0240] In contrast, an "immunoglobulin single variable domain" is capable of specifically binding to an epitope of an antigen without the need for pairing with another immunoglobulin variable domain. The binding site of an "immunoglobulin single variable domain" is formed by a single VH, a single VHH, or a single VL domain. Thus, an "immunoglobulin single variable domain" can be a light chain variable domain sequence (e.g., a VL sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH sequence or a VHH sequence) or a suitable fragment thereof; as long as it is capable of forming a single antigen binding unit; that is, a functional antigen binding unit essentially consisting of an "immunoglobulin single variable domain", such that the single antigen binding domain does not need to interact with another variable domain to form a functional antigen binding unit.

[0241] An "immunoglobulin single variable domain" may, for example, be a heavy chain "immunoglobulin single variable domain," such as VH, VHH, including camelized VH or humanized VHH. In one embodiment, it is a VHH, including camelized VH or humanized VHH. A heavy chain "immunoglobulin single variable domain" may be derived from a conventional four-chain antibody or a heavy chain antibody. For example, an "immunoglobulin single variable domain" may be a single domain antibody (or an amino acid sequence suitable for use as a single domain antibody), as defined herein, and including but not limited to VHH); other single variable domains, or any suitable fragment thereof.

[0242] In particular, an "immunoglobulin single variable domain" may be (such as a VHH, including a humanized VHH or a camelized VH) or a suitable fragment thereof. A "VHH domain", also referred to as a VHH, a VHH antibody fragment, and a VHH antibody, was initially described as an antigen-binding immunoglobulin variable domain of a "heavy chain antibody" (i.e., an "antibody without a light chain"). The term "VHH domain" is selected to distinguish these variable domains from the heavy chain variable domains present in conventional 4-chain antibodies (referred to herein as "VH domains") and the light chain variable domains present in conventional 4-chain antibodies (referred to herein as "VL domains"). For further description of VHH, reference is made to Muyldermans' review article (Reviews in Molecular Biotechnology 74:277-302, 2001).

[0243] Typically, immunoglobulin production involves immunizing an experimental animal, fusing immunoglobulin-producing cells to produce hybridomas, and screening for the desired specificity. Alternatively, immunoglobulins can be produced by screening immune or synthetic libraries, such as by phage display.

[0244] The generation of immunoglobulin sequences (such as) has been extensively described in various publications, among which WO 94 / 04678, Hamers-Casterman et al. 1993 (Nature 363:446-448, 1993) and Muyldermans et al. 2001 (Reviews in Molecular Biotechnology 74:277-302, 2001) can be cited as examples. In these methods, camelids are immunized with a target antigen in order to induce an immune response against the target antigen.

[0245] In these cases, the generation of antibodies requires purified antigen for immunization and / or screening. Antigens can be purified from natural sources or during recombinant production.

[0246] Immunization and / or screening of immunoglobulin sequences can be performed using peptide fragments of such antigens.

[0247] The present invention can use immunoglobulin sequences from different sources, including mouse, rat, rabbit, donkey, human, and camelid immunoglobulin sequences. The present invention also includes fully human sequences, humanized sequences, or chimeric sequences. For example, the present invention includes camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camelized domain antibodies. In addition, the present technology also uses fused immunoglobulin sequences, for example to form multivalent and / or multispecific constructs, and immunoglobulin sequences containing tags or other functional moieties (e.g., toxins, labels, radiochemicals, etc.), which can be derived from the immunoglobulin sequences of the present technology.

[0248] The structure of an "immunoglobulin single variable domain" sequence can be considered to be composed of four framework regions ("FR"), which are referred to in the art and herein as "Framework Region 1" ("FR1"); "Framework Region 2" ("FR2"); "Framework Region 3" ("FR3"); and "Framework Region 4" ("FR4"), respectively; the framework regions are interrupted by three complementarity determining regions ("CDRs"), which are referred to in the art and herein as "Complementarity Determining Region 1" ("CDR1"); "Complementarity Determining Region 2" ("CDR2"); and "Complementarity Determining Region 3" ("CDR3").

[0249] The determination of the CDR regions can be carried out according to different methods. In such immunoglobulin sequences, the framework sequences may be any suitable framework sequences, and examples of suitable framework sequences will be clear to the skilled person, for example based on standard handbooks and the further disclosures and prior art mentioned herein.

[0250] The framework sequence is an immunoglobulin framework sequence or a suitable combination of framework sequences derived from immunoglobulin framework sequences, for example, by humanization or camelization. For example, the framework sequence can be a framework sequence derived from a light chain variable domain (e.g., a VL sequence) and / or a heavy chain variable domain (e.g., a VH sequence or a VHH sequence). On the one hand, the framework sequence is a framework sequence derived from a VHH sequence, wherein the framework sequence can be optionally partially or fully humanized; or it is a conventional VH sequence (as defined herein) that has been camelized.

[0251] Likewise, as generally described herein for immunoglobulin sequences, any of the foregoing suitable fragments or combinations of fragments may also be used, such as fragments containing one or more CDR sequences, suitably flanked by and / or connected via one or more framework sequences; e.g., in the same order as these CDR and framework sequences may appear in the full-size immunoglobulin sequence from which the fragment is derived.

[0252] However, it should be noted that the present technology is not limited as to the origin of the "immunoglobulin single variable domain" sequence or the origin of the nucleotide sequence used to express the "immunoglobulin single variable domain" sequence, nor as to the manner in which the "immunoglobulin single variable domain" sequence or nucleotide sequence is generated or obtained or has been generated or obtained. Thus, the "immunoglobulin single variable domain" sequence can be a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence. In specific but non-limiting aspects, the "immunoglobulin single variable domain" sequence is a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence, including but not limited to "humanized" (as defined herein) immunoglobulin sequences (such as partially or fully humanized mouse or rabbit immunoglobulin sequences, and in particular partially or fully humanized VHH sequences), "camelized" (as defined herein) immunoglobulin sequences.

[0253] Similarly, the nucleotide sequence may be a naturally occurring nucleotide sequence or a synthetic or semi-synthetic sequence and may be, for example, a sequence isolated by PCR from a suitable naturally occurring template, such as DNA or RNA isolated from a cell, a nucleotide sequence that has been isolated from a library (and in particular, an expression library), a nucleotide sequence that has been prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known per se, such as mismatch PCR), a nucleotide sequence that has been prepared by PCR using overlapping primers, or a nucleotide sequence that has been prepared using DNA synthesis techniques known per se.

[0254] References to "CD33" herein include any recombinant or naturally occurring form of CD33, or variants or homologs thereof that maintain CD33 activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to CD33). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity over the entire sequence or a partial sequence (e.g., a 50, 100, 150 or 200 consecutive amino acid portion) compared to the naturally occurring CD33 protein.

[0255] " CD123 " mentioned herein includes any recombinant or naturally occurring form of CD123, or its variant or homologue (such as compared to CD123 at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity) maintaining CD123 activity. In some aspects, variant or homologue have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity in complete sequence or partial sequence (such as 50,100,150 or 200 continuous amino acid portions) compared to naturally occurring CD123 protein.

[0256] In another specific but non-limiting aspect, the amino acid sequence of the invention is an immunoglobulin sequence. Specifically, but not limiting, the amino acid sequence of the invention can be an amino acid sequence consisting essentially of four framework regions (FR1-FR4, respectively) and three complementarity determining regions (CDR1-CDR3, respectively); or any suitable fragment of an amino acid sequence that still binds to a specific epitope on CD33 or CD123.

[0257] In such amino acid sequences of the invention, the framework sequence may be any suitable framework sequence, examples of which will be clear to the skilled person (e.g. based on standard manuals and the prior art further described and mentioned herein).

[0258] The framework sequence is preferably an immunoglobulin framework sequence or a framework sequence (an appropriate combination of) which has been derived from an immunoglobulin framework sequence (e.g., by sequence optimization, such as humanization or camelization). For example, the framework sequence may be a framework derived from a light chain variable domain and / or derived from heavy chain variable domains.

[0259] The amino acids of the present invention are single domain antibodies having the following (general) structure, listed as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1-4 refer to the framework regions and CDR1-3 are the complementarity determining regions.

[0260] As used herein, "single-domain antibodies" (sdAbs, also referred to as nanobodies or VHHs by 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 that have only heavy chains (which naturally do not contain light chains), single-domain antibodies derived from conventional antibodies, and engineered antibodies.

[0261] 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 species of the Camelidae family (e.g., camels, dromedaries, llamas and guanacos). Like complete antibodies, single domain antibodies are capable of selectively binding to specific antigens. Single domain antibodies can contain only the variable domain of an immunoglobulin chain, which has CDR1, CDR2 and CDR3 and a framework region.

[0262] As used herein, the term "Fc fusion antibody" refers to a novel protein produced by fusing the Fc segment of an antibody of interest with a functional protein molecule having biological activity using genetic engineering technology.

[0263] Fc amino acid numbering follows the Kabat numbering. Taking the Fc region of wild-type human IgG as an example, "Kabat numbering" refers to the numbering system described by Kabat et al., which is recorded in the U.S. Department of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). The specific numbering is shown in Figure 19, where amino acids 221-227 represent the hinge domain, amino acids 228-340 represent the CH2 domain of the second constant region of the heavy chain, and amino acids 341-447 represent the CH3 domain of the third constant region of the heavy chain. In one embodiment, the wild-type human Fc region involved in this application is selected from IgG1, and the mutation is based on the mutation of IgG1.

[0264] As used herein, an Fc region can have "effector functions" that are responsible for activating or attenuating a biological activity (e.g., in a subject). Examples of effector functions include, but are not limited to, complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR); and the like. Such effector functions require the Fc region to be associated with a binding domain (e.g., an antibody variable domain) and can be assayed using a variety of assays (e.g., Fc binding assays, ADCC assays, CDC assays, etc.).

[0265] With respect to amino acid sequences, the skilled artisan will recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that alter, add, or delete a single amino acid or a small number of amino acids in the encoded sequence are "conservatively modified variants," where such alterations result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitutions that provide functionally similar amino acids are well known in the art.

[0266] Conservative amino acid replacement is the replacement of an amino acid residue by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Conservative modifications can be selected, for example, based on the similarity of polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved.

[0267] 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 by transplanting the complementarity determining regions (CDR1-3 sequences) of a target antibody into the variable region of a human antibody, or by subjecting the target antibody to amino acid mutations based on the characteristics of the human antibody framework region (FR1-4). Humanized antibodies can be produced synthetically or by site-directed mutagenesis.

[0268] 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.

[0269] The term "tumor" refers to all types of cancers, neoplasms or malignancies found in mammals, including leukemias, lymphomas, melanomas, neuroendocrine tumors, carcinomas and sarcomas. Examples of cancers that can be treated with the antibodies and pharmaceutical compositions provided herein include lymphomas (such as mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginal zone lymphoma, Burkitt's lymphoma), sarcomas, bladder cancer, bone cancer, brain tumors, cervical cancer, colon cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, myeloma, thyroid cancer, leukemia, prostate cancer, breast cancer, ovarian cancer, pancreatic cancer, liver cancer (such as hepatocellular carcinoma), lung cancer (such as non-small cell lung cancer, squamous cell lung cancer), and leukemia. carcinoma, adenocarcinoma, large cell lung cancer, small cell lung cancer, carcinoid, sarcoma), glioblastoma multiforme, glioma, melanoma, prostate cancer, castration-resistant prostate cancer, breast cancer, triple-negative breast cancer, glioblastoma, ovarian cancer, lung cancer, squamous cell carcinoma (e.g., of the head, neck, or esophagus), colorectal cancer, leukemia (e.g., lymphoblastic leukemia, chronic lymphocytic leukemia, hairy cell leukemia), acute myeloid leukemia, lymphoma, B-cell lymphoma, or multiple myeloma. Additional examples include thyroid cancer, endocrine system cancer, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, esophageal cancer, liver cancer, kidney cancer, lung cancer, non-small cell lung cancer, melanoma, mesothelioma, ovarian cancer, sarcoma, stomach cancer, uterine cancer or medulloblastoma, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, essential thrombocythemia, essential macroglobulinemia, primary brain tumor, cancer, malignant pancreatic insulinoma (malignant pancreatic insulinoma), pancreatic islet cell carcinoma, ... insulanoma), malignant carcinoid tumor, urinary bladder cancer, precancerous skin lesions, testicular cancer, lymphoma, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenocortical carcinoma, endocrine or exocrine pancreatic tumors, medullary thyroid cancer, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma, Paget's disease of the nipple, phyllodes tumor of the breast, lobular carcinoma, ductal carcinoma, pancreatic stellate cell carcinoma, hepatic stellate cell carcinoma, or prostate cancer.

[0270] A modified first variant Fc region polypeptide or a modified second variant Fc region polypeptide refers to: in certain embodiments, one or more additional amino acid modifications can be introduced into the Fc region of a human IgG antibody, thereby generating an Fc region variant. The variant Fc region polypeptide can be derived from a human IgG Fc region sequence (e.g., an Fc region of human IgG1, IgG2, IgG3, or IgG4), wherein the human IgG Fc region sequence comprises an amino acid modification (e.g., a substitution / mutation) at one or more amino acid positions.

[0271] This patent uses genetic engineering technology to prepare target proteins and truncated forms of target proteins, and then immunizes the obtained antigen proteins into Alxa Bactrian camels in Inner Mongolia. After multiple immunizations, peripheral blood lymphocytes or spleen cells of the camels are obtained. Through genetic engineering, the camel-derived antibody variable region coding sequence is recombined into a phage display vector. 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, autoimmune and inflammatory diseases are further tested.

[0272] The present application is to prepare a target protein and a truncated form of the target protein through genetic engineering technology, and then immunize the obtained antigen protein with Alxa Bactrian camels in Inner Mongolia. After multiple immunizations, peripheral blood lymphocytes or spleen cells of the camel are obtained, and the camel-derived antibody variable region coding sequence is recombined into a phage display vector through genetic engineering. 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, autoimmune and inflammatory diseases are further tested.

[0273] The above technical solution is now broken down into details and described in the form of specific embodiments:

[0274] Example 1: Preparation of human CD33 protein:

[0275] The human recombinant extracellular domain protein used in this patent was obtained through the company's own expression and purification. The expression vector design scheme for the human recombinant CD33 protein is as follows:

[0276] (1) The coding sequence of CD33 was retrieved from NCBI and its accession number is NM_001772.3. The amino acid sequence generated by this sequence is accession number NP_001763.3.

[0277] (2) The nucleotide sequence encoding the amino acids 1 to 259 aa of CD33 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 extensively extracted to remove endotoxins and transfected into suspension 293F cells for target protein expression and purification. The purity reached over 90%, meeting animal immunization requirements.

[0278] Example 2: Construction of a single domain antibody library against CD33 protein:

[0279] One mg of the purified human recombinant CD33 protein obtained in the above example was mixed with an equal volume of Freund's complete adjuvant and used to immunize a Bactrian camel from Alxa, Inner Mongolia. Immunizations were repeated weekly for a total of seven times. The first immunization used Freund's complete adjuvant, and the dose was twice that of subsequent immunizations. The interval between the first and second immunizations was 10 days. Except for the first immunization, the remaining six immunizations were performed using 1 mg of CD33 protein mixed with an equal volume of Freund's incomplete adjuvant. This immunization procedure was designed to stimulate the camel to produce antibodies against the CD33 protein.

[0280] After the animals were immunized, 150 mL of peripheral blood lymphocytes were collected and RNA was extracted from the cells. The extracted total RNA was used to synthesize cDNA, and the VHH (antibody heavy chain variable region) was amplified using the cDNA as a template through a nested PCR reaction.

[0281] The pMECS vector and VHH fragment were then 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 approximately 1×10 9 At the same time, the correct insertion rate of the target fragment in the library was detected by colony PCR identification.

[0282] 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%.

[0283] Example 3: Screening of single domain antibodies against CD33 protein:

[0284] 200 μL of the recombinant TG1 cells described in the above example were cultured in 2×TY medium. During this period, 40 μL of helper phage VCSM13 was added to infect the TG1 cells and cultured overnight to amplify the phage. The next day, the phages were precipitated with PEG / NaCl and the amplified phages were collected by centrifugation.

[0285] 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 also set up. The next day, 200 μL of 3% skim milk was added and the plate was blocked at room temperature for 2 hours. After blocking, 100 μL of amplified phage library (approximately 2 × 10 11 phage particles) at room temperature for 1 hour; after 1 hour, the cells were washed 15 times with PBS + 0.05% Tween-20 to remove unbound phage.

[0286] Phages specifically bound to the CD33 protein were dissociated using 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 enrich the phages.

[0287] When the enrichment multiple reaches more than 10 times, the enrichment effect is shown in Figure 1.

[0288] In Figure 1, P / N = the number of monoclonal bacteria grown after phage eluted from the positive wells in bio-panning infected with TG1 bacteria / the number of monoclonal bacteria grown after phage eluted from the positive wells infected with TG1 bacteria. This parameter will gradually increase after enrichment occurs; I / E = the total amount of phage added to the positive wells in each round of bio-panning / the total amount of phage eluted from the positive wells in each round of bio-panning. This parameter will gradually approach 1 after enrichment occurs.

[0289] Example 4: Screening of CD33-specific positive clones using phage enzyme-linked immunosorbent assay (ELISA)

[0290] Two rounds of screening for anti-CD33 single domain antibodies were performed according to the screening method described in the above example. The phage enrichment factor for anti-CD33 protein reached 10 or greater. After the screening, 384 single colonies were selected from the positive clones obtained and inoculated into 96-deep-well plates in 2×TY medium containing 100 μg / mL ampicillin. A blank control was set up. After culture at 37°C to the logarithmic phase, IPTG was added to a final concentration of 1 mM and cultured overnight at 28°C.

[0291] Crude antibody was obtained using the osmotic swelling method. CD33 recombinant protein was released into 100 mM NaHCO₃ (pH 8.3), and 100 μg of protein was coated on an ELISA plate overnight at 4°C. 100 μL of the crude antibody extract was transferred to the antigen-added ELISA plate and incubated at room temperature for 1 hour. Unbound antibody was washed with PBST, and 100 μL of a 1:2000 diluted Mouse Anti-HA tag Antibody (HRP) (Thermo Fisher) was added and incubated at room temperature for 1 hour. Unbound antibody was washed with PBST, and horseradish peroxidase colorimetric solution was added. After incubation at 37°C for 15 minutes, stop solution was added, and absorbance was read at 450 nm on a microplate reader.

[0292] 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 into LB medium containing 100 μg / mL ampicillin to extract the plasmid and perform sequencing.

[0293] 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 1B1 and 1B10 specific for CD33 protein were obtained.

[0294] The amino acid sequence of the antibody is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, 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.

[0295] The amino acid sequences of the single-domain antibodies 1B1 and 1B10 are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and their nucleotide sequences are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0296] The single-domain antibody of 1B10 was modified in the CDR region to obtain 1B10-V4, whose amino acid sequence is shown in SEQ ID NO: 3. 1B10-V4 was then humanized in the FR region to obtain 1B10-V4-F57, whose amino acid sequence is shown in SEQ ID NO: 4.

[0297] The CDR sequences of the four single-domain antibodies are shown in Tables 1 and 2 , and the protein sequences and amino acid sequences of the four single-domain antibodies are shown in Tables 3 and 4 , respectively.

[0298] Table 1: CDR sequences of four single-domain antibodies

[0299] Table 2: FR sequences of four single-domain antibodies

[0300] Table 3: Amino acid sequences of four single domain antibodies

[0301] Table 4: Nucleotide sequences of four single domain antibodies

[0302] Example 5: Purification and expression of specific single domain antibodies against CD33 protein in host bacteria Escherichia coli

[0303] The plasmids (pMECS-VHH) of the different clones obtained by sequencing analysis in Example 4 were electroporated into Escherichia coli HB2151 and plated on LB+amp+glucose plates containing ampicillin and glucose, and cultured at 37°C overnight. Single colonies were selected and inoculated into 5 mL of LB culture medium containing ampicillin and cultured in a shaker at 37°C overnight.

[0304] Inoculate 1 mL of overnight culture into 330 mL of TB medium and culture at 37°C with a shaker. When the OD600nm value reaches 0.6-0.9, add 1 M IPTG and culture overnight at 28°C with a shaker. Centrifuge to collect the E. coli and obtain a crude antibody extract using the osmotic swelling method.

[0305] The antibody was purified by nickel affinity chromatography.

[0306] Example 6: Construction of a eukaryotic expression vector for an anti-CD33 single-domain antibody Fc fusion antibody

[0307] (1) Subcloning the target sequence obtained in the above examples into a eukaryotic expression vector: The antibodies screened in the above examples were subjected to Sanger sequencing to obtain their nucleotide sequences;

[0308] (2) The above nucleotide sequences (SEQ ID NOs: 5-8 and nucleotide sequences of other single-domain antibody clones whose sequences are not shown) were synthesized 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.

[0309] (3) transforming the recombinant eukaryotic expression vector constructed in step (2) into DH5α Escherichia coli, culturing and performing plasmid extraction to remove endotoxins;

[0310] (4) Sequencing the extracted plasmid;

[0311] (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-mentioned antibody is purified by the method of Example 9.

[0312] Example 7: Expression of single domain antibodies against CD33 protein in suspension ExpiCHO-S cells

[0313] (1) 3 days before transfection, 2.5×10 5 / mL cell passaging and expansion culture of ExpiCHO-S TM The calculated volume of cells was transferred to a fresh, pre-warmed 120 mL (final volume) of ExpiCHO TMThe cells were cultured in a 500 mL shake flask containing expression medium; the cell concentration reached approximately 4 × 10 6 -6×10 6 Viable cells / mL;

[0314] (2) One day before transfection, TM The cells were diluted to a concentration of 3.5 × 10 6 viable cells / mL, and cells were cultured overnight;

[0315] (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;

[0316] (4) Use fresh ExpiCHO preheated to 37℃ TM Dilute the cells to 6 × 10 6 The calculated required volume of cells was transferred to a fresh, pre-warmed 100 mL (final volume) of ExpiCHO TM in a 500 mL shake flask containing expression medium;

[0317] (5) Gently invert to mix ExpiFectamine TM CHO reagent, use 3.7mL OptiPRO TM Dilute ExpiFectamine in culture medium TM CHO reagent, swirl or mix;

[0318] (6) Use 4 mL of refrigerated OptiPRO TM Dilute 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) with culture medium and vortex to mix thoroughly;

[0319] (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 shaking the flask during the addition process;

[0320] (8) The cells were cultured with shaking at 37°C, 8% CO2, and humidified air;

[0321] (9) Add 600 μl ExpiFectamine on the first day after transfection (18-22 hours later) TM CHO Enhancer and 24mL ExpiCHO feed;

[0322] (10) Collect the supernatant approximately 8 days after transfection (cell viability is less than 70%).

[0323] Example 8: Expression of single-domain antibodies against CD33 protein in suspension 293F cells

[0324] Recombinant single domain antibody expression experimental process (taking 500mL shake flask as an example):

[0325] (1) 3 days before transfection, 2.5×10 5 For passage and expansion of 293F cells, the calculated volume of cells was transferred to a 500 mL shake flask containing 120 mL (final volume) of fresh pre-warmed OPM-293CD05 Medium. The cell concentration reached approximately 2 × 10 6 -3×10 6 viable cells / mL.

[0326] (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.

[0327] (3) Dilute the cells to 1×10 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.

[0328] (4) Dilute PEI (1 mg / mL) with 4 mL of Opti-MEM medium and mix thoroughly by vortexing or pipetting. Dilute plasmid DNA (the plasmid DNA is the eukaryotic expression vector for the Fc fusion antibody of the anti-CD33 single-domain antibody prepared in Example 6) with 4 mL of Opti-MEM medium, mix thoroughly by vortexing, and filter through a 0.22 μm filter. Incubate at room temperature for 5 minutes.

[0329] (5) Add the diluted PEI reagent to the diluted DNA and mix thoroughly 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 shaking the flask during the addition process.

[0330] (6) The cells were cultured at 37°C, 5% CO2, and shaking at 120 rpm.

[0331] (7) Add 5 mL of OPM-CHO PFF05 feed at 24 hours and 72 hours after transfection.

[0332] (8) Collect the supernatant approximately 7 days after transfection (cell viability is less than 70%).

[0333] Example 9: Purification of single domain antibodies against CD33 protein

[0334] (1) The protein expression supernatant obtained in the above example was filtered through a 0.45 μm disposable filter to remove insoluble impurities;

[0335] (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;

[0336] (3) The filtrate is passed through a Protein A prepacked column at a flow rate of 1 mL / min. During this step, the target protein in the filtrate will bind to the filler.

[0337] (4) Wash the impurity proteins bound to the column with low salt (1× PBS) and high salt buffer (10× PBS);

[0338] (5) Elute the target protein bound to the column using glycine-HCl buffer at pH 3.0;

[0339] (6) The eluate was quickly added to a pH 9.0 Tris-HCl solution for neutralization;

[0340] (7) The neutralized protein solution was dialyzed and analyzed by SDS-PAGE to confirm that the protein purity was above 95% and the concentration was above 0.5 mg / mL, and then stored at low temperature for future use.

[0341] Example 10: Construction of a eukaryotic expression vector for a single-domain antibody

[0342] The single-domain antibody target vector RJK-V4-hFC1 used in this example was modified by our company based on the Invitrogen commercial vector pCDNA3.4, by fusing the Fc region of the human IgG1 heavy chain coding sequence. This vector contains the hinge region (CH2) and CH3 region of the IgG1 heavy chain. The specific modification scheme is as follows:

[0343] (1) Select the restriction enzyme sites XbaI and AgeI on pcDNA3.4;

[0344] (2) A multiple cloning site (MCS) and a 6×His tag were introduced at the 5′ and 3′ ends of the Fc fragment coding sequence, respectively, by overlapping PCR;

[0345] (3) amplifying the above fragment by PCR using a pair of primers with XbaI and AgeI restriction sites, respectively;

[0346] (4) using restriction endonucleases XbaI and AgeI to digest the recombinant DNA fragments in pcDNA3.4 and (3), respectively;

[0347] (5) The digested vector and the inserted fragment were ligated with T4 ligase, and the ligated product was transformed into Escherichia coli, amplified, and sequenced to obtain a recombinant plasmid.

[0348] Example 11: Expression and purification of tool antibody (Tab) targeting human CD33

[0349] Tab1 used in the examples of the present invention is gemtuzumab, and Tab3 is lintuzumab. Their sequences were searched at IMGT, and the resulting sequences were codon-optimized for mammalian cell expression systems at General Biosystems (Anhui) Co., Ltd. and cloned into the pcDNA3.1 vector. After resistance screening, plasmid-positive bacteria were selected for amplification, and the 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 transiently expressed using PEI in 293F cells (culture medium: FreeStyle 293 Expression medium, Thermo, Cat#12338026+F-68, Thermo, Cat#24040032); 5% volume of 10% Peptone (Sigma, Cat#P0521-100G) was added 6 to 24 hours after transfection and cultured at 8% CO2 and 130 rpm for approximately 7 to 8 days; when the cell viability dropped to 50%, the expression supernatant was collected and purified using a Protein A (GE, Cat#17-5438-02) gravity column; after dialysis against PBS, the concentration was determined using Nanodrop, the purity was identified using SEC, and the binding ability was verified by indirect ELISA.

[0350] The concentration of Tab1 and Tab3 obtained by this method is not less than 2 mg / ml and the purity is greater than 95%.

[0351] Example 12: Antibody antigen binding dose-effect curve determination

[0352] In this example, the antigen binding dose-response curve of the antibody was determined using a standard enzyme-linked immunosorbent assay (ELISA) procedure. The steps are as follows:

[0353] (1) Coat with 50 μL of 1 μg / mL CD33 protein (CD33 protein was purchased from Beijing Biopsies Biotechnology Co., Ltd., Catalog #CD3-H5226) at 4°C overnight.

[0354] (2) Wash the plate; add 200 μL of 5% milk and block at 37°C for 2 hours.

[0355] (3) Dilute the VHH-hFc to 2 μg / mL, and then dilute the antibody 5-fold to a total of 8 concentration gradients. The VHH-hFc here is the Fc fusion antibody of the single-domain antibody against CD33 protein prepared in Example 8 (expressed in 293F cells) and purified in Example 9. In addition, hIgG, Tab1, and Tab3 controls were set up respectively; Tab1 and Tab3 were prepared in the above examples; hIgG refers to the isotype control, an immunoglobulin molecule that does not bind to any target, and is purchased commercially, and the same applies to the following.

[0356] (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.

[0357] (5) Wash the plate; add 50 μL of HRP-Goat anti hIgG secondary antibody and incubate at 37°C for 30 minutes.

[0358] (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.

[0359] (7) Add 50 μL of stop solution (1N HCl) and read the result using a microplate reader.

[0360] (8) Draw the curve and calculate EC 50 , as shown in Figures 2 to 4.

[0361] As shown in Figures 2 to 4 , the EC50 values ​​of the 1B1-hFc1 and 1B10-hFc1 antibodies of the present invention are comparable to those of Tab1 and Tab3, indicating that they have excellent antibody-antigen binding activity.

[0362] Example 13: Determination of ADCC Effects of Antibodies 1B1-hFc, 1B10-hFc, 1B10-V4-hFc, and 1B10-V4-F57-hFc

[0363] The ADCC effect of antibodies 1B1-hFc and 1B10-hFc was determined using a reporter gene assay. The steps are as follows:

[0364] (1) After thawing, cells (CHO-K1-CD33) at passage 3-4 were collected and plated into 96-well plates at a density of 10,000 cells per well. The method for constructing a tumor cell (CHO-K1-CD33) cell line was as described in the above example.

[0365] (2) Tab3, 1B1-hFc, 1B10-hFc, 1B10-V4-hFc, and 1B10-V4-F57-hFc samples were prepared into solutions with a maximum concentration of 10 μg / mL and subjected to 10-fold gradient dilution to obtain 7 concentrations; 1B1-hFc herein refers to the Fc fusion antibody of the single-domain antibody of 1B1 prepared in Example 9 (expressed in 293F cells) and purified by the method of Example 9. 1B10-hFc refers to the Fc fusion antibody of the single-domain antibody of 1B10 prepared in Example 9 (expressed in 293F cells) and purified by the method of Example 9. Tab3 was prepared by the method of the above example.

[0366] (3) Add the gradient diluted antibody solution to the cell culture wells in equal volumes of the cell suspension;

[0367] (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 density of 30,000 cells per well;

[0368] (5) After 6 hours of incubation, cell killing was detected using the One-Glo kit and luminescence was read;

[0369] (6) Calculate the fold of induction = (sample-BG) / (E / T-BG);

[0370] 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. As shown in Figures 5 to 9.

[0371] As shown in Figures 5 to 9 , the EC50 values ​​of the 1B1-hFc1, 1B10-hFc1, 1B10-V4-hFc and 1B10-V4-F57-hFc antibodies of the present invention are comparable to that of Tab3, indicating excellent ADCC activity.

[0372] Example 14: Construction of CHO-K1-CD33 cell line

[0373] The CDS region sequence information of CD33 (accession number: NP_001763.3) was obtained from the NCBI database. 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. After lentiviral packaging, the CMV-CD33-EF1a-Puromycin gene was introduced into the CHO-K1 genome using the lentiviral infection method. Puromycin resistance screening and monoclonal screening were used to obtain a CHO-K1-CD33 stable cell line stably expressing CD33 membrane protein.

[0374] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

[0375] Example 15: Preparation of human CD123 protein:

[0376] The expression vector design scheme for the human recombinant CD123 protein used in this patent is as follows:

[0377] The human recombinant CD123 protein was expressed and purified by the company itself. The expression vector design of the human recombinant CD123 protein is as follows:

[0378] (1) The coding sequence of CD123 was retrieved from NCBI and its accession number is NM_006023.2. The amino acid sequence generated by this sequence is accession number NP_006014.2.

[0379] (2) The nucleotide sequence encoding the amino acids of CD123, 19 to 300 aa, 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 suspension 293F cells for target protein expression and purification. The purity reached over 90%, meeting animal immunization requirements.

[0380] Example 16: Construction of a single domain antibody library against CD123 protein:

[0381] One mg of the purified human recombinant CD33 protein obtained in the above example was mixed with an equal volume of Freund's complete adjuvant and used to immunize a Bactrian camel from Alxa, Inner Mongolia. Immunizations were repeated weekly for a total of seven times. The first immunization used Freund's complete adjuvant, and the dose was twice that of subsequent immunizations. The interval between the first and second immunizations was 10 days. Except for the first immunization, the remaining six immunizations were performed using 1 mg of CD33 protein mixed with an equal volume of Freund's incomplete adjuvant. This immunization procedure was designed to stimulate the camel to produce antibodies against the CD33 protein.

[0382] After the animals were immunized, 150 mL of peripheral blood lymphocytes were collected and RNA was extracted from the cells. The extracted total RNA was used to synthesize cDNA, and the VHH (antibody heavy chain variable region) was amplified using the cDNA as a template through a nested PCR reaction.

[0383] The pMECS vector and VHH fragment were then 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 CD123 protein and measure the library capacity. The library capacity was approximately 1×10 9 At the same time, the correct insertion rate of the target fragment in the library was detected by colony PCR identification.

[0384] 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%.

[0385] Example 17: Screening of single domain antibodies against CD123 protein:

[0386] 200 μL of the recombinant TG1 cells described in the above example were cultured in 2×TY medium. During this period, 40 μL of helper phage VCSM13 was added to infect the TG1 cells and cultured overnight to amplify the phage. The next day, the phages were precipitated with PEG / NaCl and the amplified phages were collected by centrifugation.

[0387] 500 μg of CD123 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 also set up. The next day, 200 μL of 3% skim milk was added and the plate was blocked at room temperature for 2 hours. After blocking, 100 μL of amplified phage library (approximately 2 × 10 11 phage particles) at room temperature for 1 hour; after 1 hour, the cells were washed 15 times with PBS + 0.05% Tween-20 to remove unbound phage.

[0388] Phages specifically bound to the CD123 protein were dissociated using 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 enrich the phages.

[0389] When the enrichment multiple reaches more than 10 times, the enrichment effect is shown in FIG10 .

[0390] In Figure 10, P / N = the number of monoclonal bacteria grown after phages eluted from the positive wells in bio-panning infected with TG1 bacteria / the number of monoclonal bacteria grown after phages eluted from the positive wells infected with TG1 bacteria. This parameter will gradually increase after enrichment occurs; I / E = the total amount of phages added to the positive wells in each round of bio-panning / the total amount of phages eluted from the positive wells in each round of bio-panning. This parameter will gradually approach 1 after enrichment occurs.

[0391] Example 18: Screening of specific positive clones for CD123 using phage enzyme-linked immunosorbent assay (ELISA):

[0392] According to the screening method in the above embodiment, two rounds of screening were performed for the single-domain antibody against CD123 protein. The phage enrichment factor of the anti-CD123 protein reached more than 10. After the screening, 384 single colonies were selected from the positive clones obtained by screening and inoculated into 96 deep-well plates in 2 × TY medium containing 100 μg / mL ampicillin. A blank control was set and cultured at 37 ° C to the logarithmic phase. After that, IPTG was added at a final concentration of 1 mM and cultured at 28 ° C overnight.

[0393] Crude antibody was obtained using the osmotic swelling method; CD123 recombinant protein was released into 100mM NaHCO3, pH 8.3, and 100μg of 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, Thermo Fisher) 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 a microplate reader.

[0394] 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 transplanted into LB medium containing 100 μg / mL ampicillin to extract the plasmid and perform sequencing.

[0395] 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 1A2, 1A5, 1A8, 1B5, 2B1, and 2B6 specific for the CD123 protein were obtained.

[0396] The amino acid sequence of the antibody is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, 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.

[0397] The single-domain antibody 2B6 was subjected to CDR3 modification and FR region humanization to obtain 2B6-V162.

[0398] The humanization method is completed by high-throughput screening of an antibody framework region mutation library constructed based on big data analysis results. The detailed steps are as follows:

[0399] (1) Sequence analysis of human / camelid antibody data: amino acid preference analysis was performed on 13,873 Nb (Human) sequences downloaded in batches from the NCBI website, and amino acid preference analysis was performed on 2,000 nanobody sequences of our company to obtain amino-terminal ratio data for each site in the framework region;

[0400] (2) Comprehensive weighted analysis of human and camel sources: The above-mentioned source / camel-derived antibody sequences were uniformly numbered according to the IMGT numbering rules and corresponded one to one. Combined with the amino acid ratio analysis results of the above two species, a weighted analysis was performed according to the weight of 90% human and 10% camel, and the weighted ratio of amino acids at each site was calculated and sorted from high to low. According to the final weighted results, only amino acid species with a proportion greater than 10% were retained at a single site in the framework region, and the final weight of amino acids with a proportion greater than 10% was calculated based on the standard of the retained proportion being 1, which served as the basis for the design of the subsequent amino acid custom library.

[0401] (3) Design of amino acid custom library: For each site to be mutated, the number of amino acids that are greater than 10% is defined as n, and the ratio of the highest to lowest values ​​in the ratio greater than 10% is defined as V. The nature of the site to be mutated is determined: if V ≥ 3 and n ≤ 2, the site is considered to be a "high concentration site", otherwise it is considered to be a "medium-low concentration site". Based on this method, the custom amino acid library is divided into two "high / medium-low concentration libraries" for the construction of the amino acid custom library. The final weight in (2) above is the reference basis for the types and ratios of amino acids at the sites in the library.

[0402] (4) High-throughput screening of custom amino acid libraries:

[0403] A humanized antibody library is constructed for the antibody strain, and the constructed library is selected with the corresponding antigen to ultimately obtain antibody sequences with higher affinity and higher degree of humanization.

[0404] The CDR sequences of the seven single-domain antibodies (1A2, 1A5, 1A8, 1B5, 2B1, 2B6, and 2B6-V162) are shown in Table 5, the FR sequences and numbers of the seven single-domain antibodies are shown in Table 6, the amino acid sequences of the seven antibodies are shown in Table 7, and the nucleotide sequences of the seven antibodies are shown in Table 8.

[0405] Table 5: CDR sequences and numbers of 7 single-domain antibodies

[0406] Table 6: FR sequences and numbers of 7 single-domain antibodies

[0407] Table 7: Amino acid sequences of 7 antibodies

[0408] Table 8: Nucleotide sequences of 7 antibodies

[0409] Example 19: Purification and expression of specific single domain antibodies against CD123 protein in host bacteria Escherichia coli

[0410] The plasmids (pMECS-VHH) of the different clones obtained by sequencing analysis in the above example were electroporated into Escherichia coli HB2151 and plated on LB+amp+glucose plates containing ampicillin and glucose, and cultured at 37°C overnight. Single colonies were selected and inoculated into 5 mL of LB culture medium containing ampicillin and cultured in a shaking incubator at 37°C overnight.

[0411] Inoculate 1 mL of overnight culture into 330 mL of TB medium and culture at 37°C with a shaker. When the OD600nm value reaches 0.6-0.9, add 1 M IPTG and culture overnight at 28°C with a shaker. Centrifuge to collect the E. coli and obtain a crude antibody extract using the osmotic swelling method.

[0412] The antibody was purified by nickel affinity chromatography.

[0413] Example 20: Construction of a eukaryotic expression vector for Fc fusion antibody of an anti-CD123 single domain antibody

[0414] (1) Subcloning the target sequence obtained in the above examples into a eukaryotic expression vector: The antibodies screened in the above examples were subjected to Sanger sequencing to obtain their nucleotide sequences;

[0415] (2) The above nucleotide sequences (SEQ ID NOs: 8-14) were synthesized 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 was as described in the above examples.

[0416] (3) transforming the recombinant eukaryotic expression vector constructed in step (2) into DH5α Escherichia coli, culturing and performing plasmid extraction to remove endotoxins;

[0417] (4) Sequencing the extracted plasmid;

[0418] (5) The confirmed recombinant vector is prepared for subsequent eukaryotic cell transfection and expression. The Fc protein of VHH is expressed by the method of the above embodiment and the above antibody is purified by the method of Example 9.

[0419] Example 21: Expression of single domain antibodies against CD123 protein in suspension ExpiCHO-S cells

[0420] (1) 3 days before transfection, 2.5×10 5 / mL cell passaging and expansion culture of ExpiCHO-S TM The calculated volume of cells was transferred to a fresh, pre-warmed 120 mL (final volume) of ExpiCHO TM The cells were cultured in a 500 mL shake flask containing expression medium; the cell concentration reached approximately 4 × 10 6 -6×10 6 Viable cells / mL;

[0421] (2) One day before transfection, TM The cells were diluted to a concentration of 3.5 × 10 6 viable cells / mL, and cells were cultured overnight;

[0422] (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;

[0423] (4) Use fresh ExpiCHO preheated to 37℃ TM Dilute the cells to 6 × 10 6The calculated required volume of cells was transferred to a fresh, pre-warmed 100 mL (final volume) of ExpiCHO TM in a 500 mL shake flask containing expression medium;

[0424] (5) Gently invert to mix ExpiFectamine TM CHO reagent, use 3.7 mL OptiPRO TM Dilute ExpiFectamine in culture medium TM CHO reagent, swirl or mix;

[0425] (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-CD123 single-domain antibody prepared in Example 6) was diluted with culture medium and vortexed to mix;

[0426] (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 shaking the flask during the addition process;

[0427] (8) The cells were cultured with shaking at 37°C, 8% CO2, and humidified air;

[0428] (9) Add 600 μl ExpiFectamine on the first day after transfection (18-22 hours later) TM CHO Enhancer and 24mL ExpiCHO feed;

[0429] (10) Collect the supernatant approximately 8 days after transfection (cell viability is less than 70%).

[0430] Example 22: Expression of single domain antibodies against CD123 protein in suspension 293F cells

[0431] Recombinant single domain antibody expression experimental process (taking 500mL shake flask as an example):

[0432] (1) 3 days before transfection, 2.5×10 5 For passage and expansion of 293F cells, the calculated volume of cells was transferred to a 500 mL shake flask containing 120 mL (final volume) of fresh pre-warmed OPM-293CD05 Medium. The cell concentration reached approximately 2 × 10 6 -3×10 6 Viable cells / mL.

[0433] (2) On the day of transfection, measure the cell density and percentage of viable cells. The cell density should reach about 2×106 -3×10 6 viable cells / mL.

[0434] (3) Dilute the cells to 1×10 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.

[0435] (4) Dilute PEI (1 mg / mL) reagent with 4 mL of Opti-MEM medium and vortex or pipette to mix thoroughly; dilute plasmid DNA (the plasmid DNA is the Fc fusion antibody eukaryotic expression vector of the anti-CD123 single-domain antibody prepared in the above example) with 4 mL of Opti-MEM medium, vortex to mix thoroughly, and filter through a 0.22 μm filter. Incubate at room temperature for 5 minutes.

[0436] (5) Add the diluted PEI reagent to the diluted DNA and mix thoroughly 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 shaking the flask during the addition process.

[0437] (6) The cells were cultured at 37°C, 5% CO2, and shaking at 120 rpm.

[0438] (7) Add 5 mL of OPM-CHO PFF05 feed at 24 hours and 72 hours after transfection.

[0439] (8) Collect the supernatant approximately 7 days after transfection (cell viability is less than 70%).

[0440] Example 23: Purification of single domain antibodies against CD123 protein

[0441] (1) The protein expression supernatant obtained in Example 21 or 22 was filtered through a 0.45 μm disposable filter to remove insoluble impurities;

[0442] (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;

[0443] (3) The filtrate is passed through a Protein A prepacked column at a flow rate of 1 mL / min. During this step, the target protein in the filtrate will bind to the filler.

[0444] (4) Wash the impurity proteins bound to the column with low salt (1× PBS) and high salt buffer (1× PBS);

[0445] (5) Elute the target protein bound to the column using glycine-HCl buffer at pH 3.0;

[0446] (6) The eluate was quickly added to a pH 9.0 Tris-HCl solution for neutralization;

[0447] (7) The neutralized protein solution was dialyzed and analyzed by SDS-PAGE to confirm that the protein purity was above 95% and the concentration was above 0.5 mg / mL, and then stored at low temperature for future use.

[0448] Example 24: Construction of a eukaryotic expression vector for a single-domain antibody

[0449] The target vector RJK-V4-hFC1, a universal target vector for nanobodies used in this example, was modified by our company based on the Invitrogen commercial vector pCDNA3.4, by fusing the Fc region of the human IgG1 heavy chain coding sequence. This vector contains the hinge (CH2) and CH3 regions of the IgG1 heavy chain. The specific modification scheme is as follows:

[0450] (1) Select the restriction enzyme sites XbaI and AgeI on pcDNA3.4;

[0451] (2) A multiple cloning site (MCS) and a 6×His tag were introduced at the 5′ and 3′ ends of the Fc fragment coding sequence, respectively, by overlapping PCR;

[0452] (3) amplifying the above fragment by PCR using a pair of primers with XbaI and AgeI restriction sites, respectively;

[0453] (4) using restriction endonucleases XbaI and AgeI to digest the recombinant DNA fragments in pcDNA3.4 and (3), respectively;

[0454] (5) The digested vector and the inserted fragment were ligated with T4 ligase, and the ligated product was transformed into Escherichia coli, amplified, and sequenced to obtain a recombinant plasmid.

[0455] Example 25: Expression and purification of tool antibody (Tab) targeting human CD123

[0456] The Tab used in the examples of the present invention was prepared as follows:

[0457] Tab1, TPP-8987, sequence from US2019365916A1;

[0458] Tab2, TPP-9476, sequence from US2019365916A1;

[0459] Tab3, TPP-8988, sequence from US2019365916A1;

[0460] Tab4, talacotuzumab, sequence from IMGT.

[0461] The sequence was codon-optimized for mammalian cell expression by General Biosystems (Anhui) Co., Ltd. and cloned into the pcDNA3.1 vector. After resistance screening, plasmid-positive bacteria were selected for amplification and the plasmid was 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 transiently expressed using PEI in 293F cells (culture medium: FreeStyle 293 Expression medium, Thermo, Cat#12338026 + F-68, Thermo, Cat#24040032). 6 to 24 hours after transfection, 5% volume of 10% Peptone (Sigma, Cat#P0521-100G) was added and cultured at 8% CO2, 130 rpm for approximately 7 to 8 days. When cell viability dropped to 50%, the expression supernatant was harvested and purified using a Protein A (GE, Cat#17-5438-02) gravity column. After dialysis against PBS, the concentration was determined using Nanodrop, the purity was determined using SEC, and the binding capacity was verified by indirect ELISA.

[0462] The concentration of Tab1, Tab2, Tab3 and Tab4 obtained by the method is not less than 2 mg / ml and the purity is greater than 95%.

[0463] Example 26: Antibody Antigen Binding Dose-Effect Curve Determination

[0464] This example uses a standard enzyme-linked immunosorbent assay (ELISA) procedure, and the specific steps are as follows:

[0465] (1) Coat with 50 μL of 1 μg / mL CD123 protein (purchased from Beijing Biopsies Biotechnology Co., Ltd., catalog number: ILA-H52H6) at 4°C overnight.

[0466] (2) Wash the plate; add 200 μL of 5% milk and block at 37°C for 2 hours.

[0467] (3) Dilute VHH-hFc to 2ug / mL, and then dilute the antibody 5-fold gradient for a total of 8 concentration gradients. The VHH-hFc here is obtained by purifying the Fc fusion antibody of the single-domain antibody against CD123 protein prepared in Example 8 (expressed in 293F cells) through the above example. In addition, hIgG, Tab1, Tab2, Tab3, and Tab4 controls are also set up respectively; Tab1, Tab2, Tab3, and Tab4 are prepared by the above example; 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.

[0468] (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.

[0469] (5) Wash the plate; add 50 μL of HRP-Goat anti hIgG secondary antibody and incubate at 37°C for 30 minutes.

[0470] (6) Wash the plate several times; add 50 μL of TMB that has been restored to room temperature and react at room temperature for 15 minutes in the dark.

[0471] (7) Add 50 μL of stop solution (1N HCl) and read the result using a microplate reader.

[0472] (8) Draw the curve and calculate EC 50 , as shown in Figures 11 to 14.

[0473] As shown in Figures 11-14, the EC50 values ​​of the antibodies 1A2-hFc, 1A5-hFc, 1A8-hFc, 1B5-hFc, 2B1-hFc and 2B6-hFc of the present invention are comparable to those of Tab, demonstrating that they all have excellent antigen-binding activity.

[0474] Example 27: Reporter Method ADCC Determination of ADCC Effects of 1A5-hFc, 1A8-hFc, 1B5-hFc, 2B1-hFc, and 2B6-hFc

[0475] Here are the steps:

[0476] (1) Cells (CHO-K1-CD123) at passage 3-4 after recovery were collected and plated into 96-well plates at 10,000 cells / well; the construction process of CHO-K1-CD123 cells is shown in the above example.

[0477] (2) Tab1-Tab4, hIgG and VHH-hFc samples were prepared with a maximum concentration of 10 μg / mL solution and diluted 10-fold to obtain 7 concentrations; the VHH-hFc here was obtained by the Fc fusion antibody of the single domain antibody (1A5, 1A8, 1B5, 2B1, 2B6) against CD123 protein prepared in Example 8 (expressed in 293F cells) and purified by the method of the above example.

[0478] (3) Add the gradient diluted antibody solution to the cell culture wells in equal volumes of the cell suspension;

[0479] (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 density of 30,000 cells per well;

[0480] (5) After 6 hours of incubation, cell killing was detected using the One-Glo kit and luminescence was read;

[0481] (6) Calculate the fold of induction = (sample-BG) / (E / T-BG);

[0482] 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 The results are shown in Figures 6 and 7.

[0483] As shown in Figures 15 and 16, the EC of the 1A5-hFc, 1A8-hFc, 1B5-hFc, 2B1-hFc, and 2B6-hFc antibodies of the present invention 50 The value was comparable to that of Tab, demonstrating its excellent ADCC effect.

[0484] Example 28: Reporter method ADCC determination of the ADCC effect of 2B6-V162-hFc:

[0485] Here are the steps:

[0486] (3) The cells (CHO-K1-CD123) at passage 3-4 after recovery were collected and plated into 96-well plates at 10,000 cells / well; the construction process of CHO-K1-CD123 cells is shown in the above embodiment.

[0487] (4) Tab4, hIgG and VHH-hFc samples were prepared into a solution with a maximum concentration of 10 μg / mL and diluted 10-fold to obtain 7 concentrations; the VHH-hFc here was obtained by the Fc fusion antibody of the anti-CD123 protein single domain antibody 2B6-V162 (expressed in 293F cells) prepared in Example 8 and purified by the method of the above example.

[0488] (3) Add the gradient diluted antibody solution to the cell culture wells in equal volumes of the cell suspension;

[0489] (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 density of 30,000 cells per well;

[0490] (5) After 6 hours of incubation, cell killing was detected using the One-Glo kit and luminescence was read;

[0491] (6) Calculate the fold of induction = (sample-BG) / (E / T-BG);

[0492] 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 The results are shown in Figures 17 and 18.

[0493] As shown in Figures 17 and 18, the 2B6-V162-hFc antibody EC of the present invention 50 The value was comparable to that of Tab, demonstrating its excellent ADCC effect.

[0494] Example 29: Construction of CHO-K1-CD123 cell line

[0495] The CDS region sequence information of CD123 (accession number: NP_002174.1) was obtained from the NCBI database. The DNA sequence was selected by gene synthesis and cloned into the PHB-Puro expression vector to construct the PHB-CD123-Puro lentiviral vector plasmid. After lentiviral packaging, the CMV-CD123-EF1a-Puromycin gene was introduced into the CHO-K1 genome using the lentiviral infection method. Puromycin resistance screening and monoclonal screening were performed to obtain a CHO-K1-CD123 stable cell line stably expressing CD123 membrane protein.

[0496] Example 30: Construction of a single domain antibody library against CD33 and CD123

[0497] The steps for constructing a single domain antibody library targeting CD33 and CD123 are as follows. The methods and reagents used below are familiar to those skilled in the art:

[0498] 1. Prepare recombinant proteins of CD33 and CD123 respectively:

[0499] 1.1 Preparation of human CD33 protein:

[0500] The human recombinant extracellular domain protein used in this patent was obtained through the company's own expression and purification. The expression vector design scheme for the human recombinant CD33 protein is as follows:

[0501] (1) The coding sequence of CD33 was retrieved from NCBI and its accession number is NM_001772.3. The amino acid sequence generated by this sequence is accession number NP_001763.3.

[0502] (2) The nucleotide sequence encoding the amino acids 1 to 259 aa of CD33 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 extensively extracted to remove endotoxins and transfected into suspension 293F cells for target protein expression and purification. The purity reached over 90%, meeting animal immunization requirements.

[0503] 1.2 Preparation of CD123 recombinant protein. The expression vector design scheme for human recombinant CD123 protein is as follows:

[0504] (1) The coding sequence of CD123 was retrieved from NCBI and its accession number is NM_006023.2. The amino acid sequence generated by this sequence is accession number NP_006014.2.

[0505] (2) The nucleotide sequence encoding the amino acids of CD123, 19 to 300 aa, 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 suspension 293F cells for target protein expression and purification. The purity reached over 90%, meeting animal immunization requirements.

[0506] 2. Construct a single domain antibody library targeting CD33 and CD123 as follows:

[0507] 2.1. Human recombinant CD33 protein was mixed with an equal volume of Freund's complete adjuvant and used to immunize a Bactrian camel from Alxa, Inner Mongolia, once weekly for a total of seven immunizations. The first immunization used Freund's complete adjuvant, and the dose was twice that of subsequent immunizations. The interval between the first and second immunizations was 10 days. Except for the first immunization, the remaining six immunizations were performed using 1 mg of CD33 protein mixed with an equal volume of Freund's incomplete adjuvant. This immunization process is intended to stimulate the camel to produce antibodies against the CD33 protein.

[0508] 2.2. After immunization, 100 mL of peripheral blood lymphocytes were collected from the camels and total RNA was extracted.

[0509] 2.3. cDNA was synthesized and the variable domain (VHH) of the heavy chain antibody was amplified using nested PCR.

[0510] 2.4. Use restriction endonucleases Pst I and Not I to digest 20 μg of pMECS phage display vector and 10 μg of VHH, and ligate the digested VHH and vector.

[0511] 2.5. The ligation product was transformed into electrocompetent TG1 cells to construct a CD33 protein phage display library. PCR amplification of 30 randomly selected colonies from the library revealed that 28 clones amplified bands of the predicted size, while 2 clones amplified incorrect bands, resulting in a correct insertion rate of 28÷30×100%≈93%.

[0512] 2.6. The steps for constructing the single domain antibody library targeting CD123 were the same as those for constructing the CD33 library (same as steps 2.1 to 2.5). The results showed that the correct insertion rate reached 93%.

[0513] Example 31: Enrichment of a single domain antibody library targeting CD33 and CD123

[0514] The steps for enriching the single domain antibody library are as follows. The methods and reagents used below are familiar to those skilled in the art:

[0515] 1. Take 200 μL of recombinant TG1 cells and culture them in 2×TY medium. During this period, add 40 μL of helper phage VCSM13 to infect TG1 cells and culture them overnight to amplify phage. The next day, use PEG / NaCl to precipitate the phage and collect the amplified phage by centrifugation.

[0516] 2. The CD33 protein prepared in the above example was diluted to 5 ng / μL with 0.1 M sterile NaHCO3. 100 μL of CD33 protein was added to the positive wells and 100 μL of sterile NaHCO3 was added to the negative wells. The cells were incubated at 4°C overnight.

[0517] 3. The next day, discard the overnight coated protein, add 300 μL 1‰ PBST to the wells and wash the plate 10 times. Shake off and pat dry after each addition, then add 300 μL 3% skim milk and block at room temperature for 2 hours.

[0518] 4. After blocking, discard the skim milk, wash the plate 10 times with 300 μL 1‰ PBST, and then add 100 μL amplified phage library (about 2×10 11 phage particles) and allowed to react at room temperature for 1 hour.

[0519] 5. After 1 hour of reaction, wash five times with 300 μL 1‰ PBST to wash away unbound phages.

[0520] 6. Use trypsin at a final concentration of 0.25 mg / mL to dissociate the specifically bound phage and infect E. coli TG1 cells in the logarithmic growth phase. Incubate at 37°C for 1 hour to produce and collect phage for the next round of screening. Repeat the same screening process twice to gradually enrich.

[0521] The enrichment effect of the CD33 single-domain antibody library is shown in Figure 1. The P / N value increased from 0.9 in the first round to approximately 22 in the third round, and the I / E value increased from 250,000 in the first round to approximately 476 in the third round, demonstrating that the library has a very significant enrichment for CD33 single-domain antibodies.

[0522] The enrichment steps for the CD123 single domain antibody library are the same as those for CD123 (same as steps 1 to 6). The results are shown in Figure 10. The P / N value increased from 3.8 in the first round to approximately 138 in the second round, and the I / E value increased from 105263 in the first round to approximately 1904 in the second round, demonstrating that the library has a very significant enrichment for CD33 single domain antibodies.

[0523] In Figures 1 and 10, P / N = the number of monoclonal bacteria grown after phages eluted from the positive wells in bio-panning infected with TG1 bacteria / the number of monoclonal bacteria grown after phages eluted from the positive wells infected with TG1 bacteria. This parameter will gradually increase after enrichment occurs; I / E = the total amount of phages added to the positive wells in each round of bio-panning / the total amount of phages eluted from the positive wells in each round of bio-panning. This parameter will gradually approach 1 after enrichment occurs.

[0524] Example 32: Screening of specific positive clones using phage enzyme-linked immunosorbent assay (ELISA)

[0525] The following methods and reagents are familiar to those skilled in the art:

[0526] 1. Multiple rounds of screening for CD33 or CD123 were performed according to the above-mentioned single-domain antibody screening method. After the screening, the phage enrichment factor for CD33 or CD123 reached 10 or above (approximately 10,000). 400 single colonies were selected from the positive clones obtained from the screening and inoculated into 96-deep-well plates in TB medium containing 100 μg / mL ampicillin. A blank control was set up. After culturing at 37°C to the logarithmic phase, IPTG was added to a final concentration of 1 mM and the culture was induced at 28°C overnight. Crude antibodies were obtained by the osmotic swelling method.

[0527] 2. Dilute CD33 protein or CD123 protein into 100 mM NaHCO3 (pH 8.3) and coat 100 μg of CD33 protein in an ELISA plate at 4°C overnight. The next day, add 100 μg of BSA-Biotin protein to the plate.

[0528] 3. Transfer 100 μL of the crude antibody extract obtained in the above step to the ELISA plate with antigen added and incubate at room temperature for 1 hour.

[0529] 4. Wash away unbound antibodies with PBST, add 100 μL of 1:2000 diluted mouse anti-HA tag antibody (mouse anti-HA antibody, purchased from Thermo Fisher), and incubate at room temperature for 1 hour.

[0530] 5. Wash away unbound antibodies with PBST, add 100 μL of 1:20,000 diluted Anti-Rabbit HRP conjugate (goat anti-rabbit horseradish peroxidase-labeled antibody, purchased from Thermo Fisher), and incubate at room temperature for 1 hour.

[0531] 6. Wash away unbound antibodies with PBST, add horseradish peroxidase colorimetric solution, react at 37°C for 15 minutes, add stop solution, and read the absorbance value OD450 at a wavelength of 450 nm on a microplate reader.

[0532] 7. If the OD value of the sample well is more than 5 times greater than that of the control well, the well is determined to be a positive clone well.

[0533] 8. Transfer the cells from the positive clone wells into LB medium containing 100 μg / μL ampicillin to extract the plasmid and perform sequencing.

[0534] The gene sequences of each clone were analyzed using the sequence alignment software Vector NTI. 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 or CD123 were obtained, namely 1B10 and 2B6.

[0535] The anti-CD33 single-domain antibody 1B10 was screened, and its amino acid sequence is shown in SEQ ID NO: 2. 1B10 was modified with amino acids to obtain clone number 1B10V4-F57, and its amino acid sequence is shown in SEQ ID NO: 4.

[0536] The amino acid sequence of the screened anti-CD123 single-domain antibody 2B6 is shown in SEQ ID NO: 32. 2B6 was subjected to amino acid modification to obtain clone number 2B6-V162, whose amino acid sequence is shown in SEQ ID NO: 33.

[0537] The amino acid sequence of the antibody is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, which constitutes the entire VHH. The obtained single-domain antibody recombinant plasmid can be expressed in a prokaryotic system to ultimately obtain a single-domain antibody protein.

[0538] Example 33: Construction of eukaryotic expression vector for FC fusion antibody

[0539] The antibodies 1B10V4-F57 and 2B6-V162 in the above examples were fused to FC to prepare FC fusion antibodies, and eukaryotic vectors were constructed as follows. The methods and reagents used below are familiar to those skilled in the art:

[0540] The antibodies 1B10V4-F57 and 2B6-V162 in the above examples were respectively subjected to Sanger sequencing to obtain their nucleotide sequences (the nucleotide sequence of the anti-CD33 single domain antibody is SEQ ID NO: 72; the nucleotide sequence of the anti-CD123 single domain antibody is SEQ ID NO: 73).

[0541] Next, the above nucleotide sequence and the nucleotide sequence of the Fc variant were synthesized into the vector RJK-V4-hFC by sequence synthesis. The preparation method and specific sequence of the RJK-V4-hFC vector can be found in Application No. 201911238332.2, Publication No. CN 111018985 A, published on April 17, 2020. A plasmid containing the nucleotide sequence encoding the first full-length antibody and a plasmid containing the nucleotide sequence encoding the second full-length antibody were obtained, respectively.

[0542] The plasmid containing the nucleotide sequence encoding the first full-length antibody can be obtained by the following method:

[0543] 1. First insert the nucleotide sequence of the anti-CD33 single-domain antibody into the vector RJK-V4-hFC, and then insert the nucleotide sequence of the corresponding Fc variant (i.e., the modified first variant Fc region polypeptide); or

[0544] 2. The nucleotide sequence of the anti-CD33 single-domain antibody and the nucleotide sequence of the FC variant (ie, the modified first variant Fc region polypeptide) were inserted into the vector RJK-V4-hFC at one time.

[0545] The plasmid containing the nucleotide sequence encoding the second full-length antibody can be obtained by the following method:

[0546] 1. First insert the nucleotide sequence of the anti-CD123 single-domain antibody into the vector RJK-V4-hFC, and then insert the nucleotide sequence of the corresponding Fc variant (i.e., the modified second variant Fc region polypeptide); or

[0547] 2. Insert the nucleotide sequence of the anti-CD123 single-domain antibody and the nucleotide sequence of the FC variant (i.e., the modified second variant Fc region polypeptide) into the vector RJK-V4-hFC at one time.

[0548] In the above nucleotide sequences and the nucleotide sequences of the Fc variants, the nucleotide sequence of the anti-CD33 single domain antibody is at the 5' end of the nucleotide sequence of the first full-length antibody, the nucleotide sequence of the modified first variant Fc region is at the 3' end of the nucleotide sequence of the first full-length antibody, and the nucleotide sequence of the first full-length antibody is directly synthesized as a whole. The nucleotide sequence of the anti-CD123 single domain antibody is at the 5' end of the nucleotide sequence of the second full-length antibody, the nucleotide sequence of the modified second variant Fc region is at the 3' end of the nucleotide sequence of the second full-length antibody, and the nucleotide sequence of the second full-length antibody is directly synthesized as a whole.

[0549] The constructed recombinant eukaryotic expression vector was transformed into DH5α Escherichia coli, and the CD33 or CD123 plasmid was extracted in large quantities, respectively, to obtain a plasmid containing the nucleotide sequence encoding the first full-length antibody and a plasmid containing the nucleotide sequence encoding the second full-length antibody, respectively. Endotoxins were removed, and the extracted plasmids were sequenced and identified. The recombinant vectors that were identified were stored for subsequent eukaryotic cell transfection and expression.

[0550] Example 34: Eukaryotic Expression of Bispecific Antibodies

[0551] The steps for constructing a bispecific antibody using the Fc fusion antibody of the anti-CD33 single-domain antibody 1B10V4-F57 and the Fc fusion antibody of the anti-CD123 single-domain antibody 2B6-V162 in Example 33 are as follows:

[0552] 1. 3 days before transfection, use 2.5×10 5For passage and expansion of 293F cells, calculate the required cell volume and transfer it to a 500 mL shake flask filled with fresh pre-warmed 120 mL (final volume) of OPM-293CD05 Medium. The cell concentration reaches approximately 2 × 10 6 -3×10 6 viable cells / mL.

[0553] 2. On the day of transfection, measure the cell density and percentage of live cells. The cell density should reach about 2×10 6 -3×10 6 viable cells / mL.

[0554] 3. Dilute the cells to 1×10 with pre-warmed OPM-293CD05 Medium. 6 The required cell volume was calculated based on the number of viable cells / mL and transferred into a 500 mL shake flask containing 100 mL (final volume) of fresh pre-warmed culture medium.

[0555] 4. Dilute PEI (1 mg / mL) reagent with 4 ml of Opti-MEM medium and vortex or pipette to mix; mix CD33 plasmid and CD123 plasmid (i.e., plasmid containing the nucleotide sequence encoding the first full-length antibody and plasmid containing the nucleotide sequence encoding the second full-length antibody) in a 1:1 ratio and dilute with 4 mL of Opt-MEM medium, vortex to mix, filter through a 0.22 μm filter, and incubate at room temperature for 5 minutes.

[0556] 5. Add the diluted PEI reagent to the diluted plasmid and mix thoroughly 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 shaking the flask during the addition process.

[0557] 6. Culture the cells at 37°C, 5% CO2, and shaking at 120 rpm.

[0558] 7. Add 5 mL of OPM-CHO PFF05 feed at 24 and 72 hours after transfection.

[0559] 8. About 7 days after transfection (cell viability is less than 70%), the supernatant is collected. The supernatant contains the bispecific antibody of the present invention. A plasmid containing the nucleotide sequence encoding the first full-length antibody and a plasmid containing the nucleotide sequence encoding the second full-length antibody are respectively expressed to obtain the first full-length antibody and the second full-length antibody. During this process, the Fc region of the first full-length antibody and the Fc region of the second full-length antibody naturally pair to form a bispecific antibody. The structure of the anti-CD33 / anti-CD123 bispecific antibody produced by the above steps is shown in Figure 20.

[0560] As shown in Figure 20, the antibody provided by this scheme is a bispecific antibody that simultaneously targets CD33 antigen molecules and CD123 antigen molecules. The bispecific antibody is essentially a recombinant protein, which includes a first full-length antibody and a second full-length antibody. Each full-length antibody consists of a VHH segment (antigen binding domain), a portion of the hinge region, a CH2 domain, and a CH3 domain; a portion of the hinge region, the CH2 domain, and the CH3 domain constitute the Fc region; the VHH segment is connected to the CH2 domain via the hinge region.

[0561] Table 9 shows the amino acid sequence and numbering of the CDRs of antibodies 1B10V4-F57 and 2B6-V162, Table 10 shows the amino acid sequence and numbering of the FRs of antibodies 1B10V4-F57 and 2B6-V162, Table 11 shows the amino acid sequence and numbering of 1B10V4-F57 and 2B6-V162, Table 12 shows the nucleotide sequence and numbering of 1B10V4-F57 and 2B6-V162, Table 13 shows the full-length amino acid sequence of the anti-CD33 / anti-CD123 bispecific antibody (test molecule), Table 14 shows the full-length nucleotide sequence of the anti-CD33 / anti-CD123 bispecific antibody (test molecule), and Table 15 shows the amino acid sequences of 1B10 and 2B6.

[0562] Table 9: CDR sequences of antibodies

[0563] Table 10: FR sequences of antibodies

[0564] Table 11: Amino acid sequences of VHH

[0565] Table 12: Nucleotide sequences of VHH

[0566] Table 13: Full-length antibody sequences of anti-CD33 / anti-CD123 bispecific antibodies

[0567] Table 14: Nucleotide sequences of anti-CD33 / anti-CD123 bispecific antibodies

[0568] Table 15: Amino acid sequences of 1B10 and 2B6

[0569] The anti-CD33 / CD123 bispecific antibody obtained in this example was named as the test molecule and stored at low temperature for future use.

[0570] Example 35: Affinity kinetics detection of anti-CD33 / CD123 bispecific antibodies,

[0571] Affinity kinetics testing was performed on the test molecule prepared in Example 34 (purity of approximately 93%). The following methods and reagents are familiar to those skilled in the art. The testing steps are as follows:

[0572] (1) Preparation of SD buffer: Take an appropriate amount of Tween 20 and dissolve it in 1× PBS (pH 7.4) to make the Tween 20 content 0.02%.

[0573] (2) Preparation of antibody working solution: The test molecule was prepared at 10 μg / mL in SD buffer.

[0574] (3) Preparation of antigen working solution: The antigen (human CD33 or CD123) was first prepared into 200 nM using SD buffer, and then diluted in a 2-fold gradient, with a total of 5 concentration gradients, and a zero concentration was set in addition.

[0575] (4) Preparation of glycine stock solution (0.1 M, pH 2.0): Dissolve 7.5 g of glycine solid in an appropriate amount of deionized water, adjust the pH to 2.0 with hydrochloric acid, and then dilute to 100 mL with deionized water.

[0576] (5) Preparation of glycine working solution (0.01 M, pH 2.0): Take an appropriate amount of glycine stock solution, dilute it 10 times with deionized water, and mix well.

[0577] (6) System: Open the Data Acquisiton software in the Octet 96 and its supporting computer, use lens cleaning paper to take an appropriate amount of 75% ethanol to clean the bottom and sides of the acquisition probe, and preheat the instrument for more than 15 minutes.

[0578] (7) Sensor pre-wetting: Before the experiment begins, the sensor is soaked in SD buffer for more than 10 minutes and ready for use.

[0579] (8) Sample arrangement: Arrange the samples in a 96-well black board according to the table below, 200 μL / well, which can be adjusted according to actual conditions.

[0580] (9) Analysis procedure: As shown in Table 16 below, the time can be adjusted according to the specific experimental conditions.

[0581] Table 16 Analytical procedures

[0582] (10) After the analysis is completed, turn off the instrument and computer.

[0583] (11) Data Analysis: KD, Kon and other related constants were calculated using Data Analysis software. The results are shown in Tables 17 and 18.

[0584] Table 17 Affinity data for human CD33

[0585] Table 18 Affinity data for human CD123

[0586] Example 36: Antibody activity detection experiment of test molecules

[0587] The antibody activity of the test molecule was detected using the LDH assay. The following methods and reagents are familiar to those skilled in the art. The test molecule was the anti-CD33 / CD123 bispecific antibody prepared in Example 5 (purity approximately 93%). The ADCC procedure of the LDH assay was as follows:

[0588] (1) Tumor cells (KG-1, RPMI-8226, MV-4-11, THP-1, HAL01, NALM-6, SUP-B15, and JVM-2) at passage 3-4 after recovery were collected and plated into 96-well plates at 10,000 cells per well;

[0589] (2) Prepare a solution of the test molecule with a maximum concentration of 10 μg / mL and perform a 10-fold serial dilution to obtain 7 concentrations;

[0590] (3) Add the gradient diluted antibody solution to the cell culture wells in equal volumes of the cell suspension;

[0591] (4) For sample wells and E / T wells (antibody concentration is 0), collect PBMC cells and add 250,000 cells per well to the cell culture wells at twice the volume of the target cell suspension; for MAX wells, add lysis buffer twice the volume of the target cell suspension to each well; for MIN wells, add assay buffer twice the volume of the target cell suspension to each well;

[0592] (5) After 6 h of incubation, cell killing was detected using an LDH kit and the absorbance was read;

[0593] (6) Target cell killing rate % = (sample - E / T) / (MAX - MIN);

[0594] According to the target cell killing rate and concentration, four-parameter fitting was performed in GraphPad software to calculate the EC50 concentration of ADCC mediated by each antibody.

[0595] result:

[0596] Targeting KG-1 (human acute myeloid leukemia cell line), the ADCC effect results mediated by the test molecules are shown in FIG21 .

[0597] Targeting RPMI-8226 (human multiple myeloma cell line), the ADCC effect results mediated by the test molecules are shown in FIG22 .

[0598] Targeting MV-4-11 (human acute myeloid leukemia cell line), the ADCC effect results mediated by the test molecules are shown in FIG23 .

[0599] Targeting THP-1 (human monocytic leukemia cell line), the ADCC effect results mediated by the test molecules are shown in FIG24 .

[0600] Targeting HAL-01 (human acute lymphoblastic leukemia cell line), the ADCC effect results mediated by the test molecules are shown in FIG25 .

[0601] Targeting NALM-6 (adult B-type acute lymphoblastic leukemia cell line), the ADCC effect results mediated by the test molecules are shown in Figure 26.

[0602] Targeting SUP-B15 (Ph+ acute lymphoblastic leukemia cell line), the ADCC effect results mediated by the test molecules are shown in FIG27 .

[0603] Targeting JVM-2 (a human peripheral lymphocyte cell line infected with Epstein-Barr virus), the ADCC effect results mediated by the test molecules are shown in FIG28 .

[0604] From the above results, it can be seen that the test molecules have a good ADCC effect on blood tumor cell lines such as KG-1, RPMI-8226, MV-4-11, THP-1, HAL-01, NALM-6, SUP-B15 and JVM-2.

[0605] Example 37: In vivo pharmacodynamic study of bispecific antibodies (test molecules) in KG-1 subcutaneous xenograft model

[0606] The following methods and reagents are familiar to those skilled in the art:

[0607] KG-1 human leukemia cells were used at a density of 1×10 7 0.2 ml / mouse was subcutaneously inoculated into the right back (right axilla) of 48 CB17 SCID immunodeficient mice to establish an in vivo tumor model. The tumors were kept in the right axilla until the average volume of the tumors reached about 94 mm. 3 At PG-D0, mice were randomly divided into 6 groups (designated as PG-D0) based on tumor volume and body weight. Subsequently, the six groups received intraperitoneal injections of vehicle and 10 mg / kg of the test molecule twice weekly. Tumor volume was monitored twice weekly, and tumor proliferation rate and tumor growth inhibition rate were calculated based on tumor volume.

[0608] Compound information, name: test molecule, concentration: 57.5 mg / mL, purity: 93.36%. The test molecule is the anti-CD33 / CD123 bispecific antibody prepared in Example 5.

[0609] Solvent: 25mM NaAc-Hac, pH 6.0.

[0610] The experimental methods and steps are as follows:

[0611] (1) Cell culture and collection

[0612] Human leukemia KG-1 cells were cultured in IMDM medium supplemented with 20% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C in a 5% CO2 incubator. Routine passage was performed twice a week. When cell saturation reached 80%-90% and the desired number of cells was reached, the cells were harvested, counted, and resuspended in DPBS (containing 50% Matrigel) at a density of 5 × 10 7 cells / mL.

[0613] (2) Tumor cell inoculation, enrollment, and drug administration

[0614] 0.2 mL of KG-1 tumor cells (containing 10×10 6 The cells suspension (DPBS:Matrigel=1:1) was subcutaneously inoculated on the right back (right armpit) of the mice. In the pharmacodynamics experiment, the average tumor volume reached about 94 mm on the 20th day after cell inoculation. 3 At 3 pm, the patients were randomly divided into groups and the medication was started.

[0615] (3) Preparation of test substances

[0616] The formula and specific configuration method are shown in Table 19.

[0617] Table 19: Compound preparation method

[0618] The test molecule is the anti-CD33 / CD123 bispecific antibody prepared in Example 5.

[0619] (4) Tumor measurement and experimental indicators

[0620] Tumor diameter was measured twice a week using a vernier caliper. Tumor volume was calculated using the formula: V = 0.5a × b 2 , a and b represent the long diameter and short diameter of the tumor, respectively. The anti-tumor efficacy of the test substance was evaluated by tumor proliferation rate T / C (%) or tumor growth inhibition rate TGI (%).

[0621] The relative tumor volume (RTV) was calculated based on the tumor measurement results. The calculation formula is RTV = V t / V0, where V0 is the average tumor volume measured at the time of group administration (i.e., PG-D0), Vt is the average tumor volume at a certain measurement, T RTV with C RTV Get data for the same day.

[0622] Tumor proliferation rate T / C (%): T / C% = T RTV / C RTV × 100%, where T RTV RTV for the treatment group, C RTV RTV is the vehicle control group, T RTV with C RTV Get data for the same day.

[0623] Tumor growth inhibition rate TGI (%): TGI% = (1-T RTV / C RTV )×100%, where T RTV RTV for the treatment group, C RTV RTV is the vehicle control group, T RTV with C RTV Get data for the same day.

[0624] (5) Statistical analysis

[0625] Statistical analysis included the mean and standard error (SEM) of tumor volume at each time point for each group. Statistical analysis was performed based on the data from the treatment groups after experimental administration to assess intergroup differences. One-way ANOVA was used to compare fluorescence intensity between three or more groups. If the variances were equal (F value > 0.05), Dunnett's method was used for analysis. If the variances were unequal (F value < 0.05), the Games-Howell method was used for testing. All data were analyzed using SPSS 17.0. p > 0.05 indicated no statistical difference, and p < 0.05 indicated a significant difference.

[0626] The experimental results are as follows:

[0627] (1) Animal Observation

[0628] During the entire experimental period, the animals were observed once a day, and no obvious abnormal reactions were observed in the appearance, behavioral activities, glandular secretions, and respiration of all animals.

[0629] (2) Weight changes

[0630] The body weight of the experimental animals was used as an indirect reference indicator for determining drug toxicity. The changes in animal body weight were monitored during the experiment, and all mice maintained a good body weight during the monitoring period.

[0631] (3) Tumor volume and experimental indicators

[0632] Tumor volume, tumor proliferation rate, and tumor growth inhibition rate were measured in CB17 SCID mice bearing subcutaneous xenografts of human leukemia KG-1 cells after treatment with vehicle and the test molecule. The therapeutic effects of the test drug on subcutaneous xenografts of human leukemia KG-1 cells were analyzed.

[0633] There was no obvious upward trend in tumor volume during the administration of the test molecule group, and the tumor volume increased slightly after drug withdrawal. Details are shown in Table 20, and the tumor proliferation rate results are shown in Table 21.

[0634] Table 20: Tumor volume of each group at different time points Note: a. Mean ± SEM. *One-way ANOVA analysis of variance was performed on the tumor volumes of the drug-treated group and the RJK-RT2831 vehicle group at different time points. * indicates p < 0.05, and none indicates p > 0.05.

[0635] Table 21: Effects of the test drugs on tumor proliferation rate in KG-1 human leukemia cell xenograft tumor model Note: a. Days after grouping b. Tumor proliferation rate T / C (%): Calculation formula is T / C% = T RTV / C RTV ×100%

[0636] Tumor Growth Inhibition Rate: All drug groups tested showed tumor inhibition. Compared with the vehicle group, all tested molecule groups showed significant inhibition at the tested time points (p < 0.05). See Table 22 for details.

[0637] Table 22: Tumor growth inhibition rate of the test drugs on the KG-1 human leukemia cell xenograft tumor model Note: a. Days after grouping b. Tumor growth inhibition rate TGI (%): The calculation formula is TGI% = (1-T RTV / C RTV )×100%

[0638] Compared with the data of PG-D19 (last administration), the tumor volume of the tumor-bearing mice in the vehicle control group reached 547 mm 3 Compared with the vehicle control group, the test molecule, 10 mg / kg, BIW (tumor volume = 53 mm 3 , TGI=90.02%, p<0.05) all showed significant tumor-suppressing effects.

[0639] From the above data, it can be seen that the drug continues to show significant tumor-suppressing effects after drug withdrawal.

[0640] Conclusion: Tumor growth: There was no obvious upward trend in tumor volume during the administration of the test molecule group, and the tumor volume increased slightly after drug withdrawal.

[0641] Tumor proliferation rate: The tumor proliferation rate in the test drug groups was lower than that in the vehicle group.

[0642] Tumor Growth Inhibition Rate: All drug groups tested showed tumor inhibition. Compared with the vehicle group, the test molecule group showed significant inhibition at all tested time points (p<0.05).

[0643] Example 38 In vivo efficacy of the test molecule on the human acute myeloid leukemia MV4-11 cell subcutaneous xenograft tumor CB17 SCID mouse model

[0644] The methods and reagents used below are familiar to those skilled in the art. The experimental methods and steps are as follows:

[0645] (1) Cell culture and collection

[0646] Human acute myeloid leukemia MV4-11 cells (purchased from ATCC, catalog number CRL-9591) were cultured in suspension in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C in a 5% CO2 incubator. Routine passage was performed twice a week. When the cell confluence reached 80%-90% and the cell number reached the required level, the cells were washed, collected, and resuspended in PBS (containing 50% Matrigel) at a density of 5 × 10 7 cells / mL.

[0647] (2) Tumor cell inoculation and animal administration

[0648] Aspirate 0.2mL (10×10 6 MV4-11 cell suspension (PBS:Matrigel=1:1) was subcutaneously inoculated on the right back (right armpit) of each mouse. Pharmacological experiments were performed on the 7th day after cell inoculation, when the average tumor volume reached approximately 95 mm 3 At 3 pm, the patients were randomly divided into groups and the medication was started.

[0649] (3) Preparation of test substances

[0650] The recipe and specific configuration method are shown in Table 23.

[0651] Table 23: Compound preparation method

[0652] The test molecule is the anti-CD33 / CD123 bispecific antibody prepared in Example 5.

[0653] (4) Tumor measurement and experimental indicators

[0654] The experimental indicator is to determine whether tumor growth is inhibited, delayed, or cured. Tumor diameter is measured twice weekly with a vernier caliper. Tumor volume is calculated using the formula: V = 0.5a × b², where a and b represent the major and minor diameters of the tumor, respectively.

[0655] The relative tumor volume (RTV) was calculated based on the tumor measurement results using the formula RTV = Vt / V0, where V0 is the average tumor volume measured at the time of group administration (i.e., PG-D0), and Vt is the average tumor volume at a particular measurement.

[0656] The anti-tumor efficacy of the compound was evaluated by relative tumor inhibition rate TGI (%) or relative tumor proliferation rate T / C (%). TGI (%) reflects the tumor growth inhibition rate.

[0657] Tumor proliferation rate T / C (%): T / C%=TRTV / CRTV×100%, where TRTV is the RTV of the treatment group, CRTV is the RTV of the vehicle control group, and the data of TRTV and CRTV were collected on the same day.

[0658] Tumor growth inhibition rate TGI (%): TGI% = (1-TRTV / CRTV) × 100%, where TRTV is the RTV of the treatment group, CRTV is the RTV of the vehicle control group, and TRTV and CRTV data were collected on the same day.

[0659] (5) Data processing and analysis

[0660] Statistical analysis included the mean and standard error (SEM) of tumor volume at each time point for each group. Intergroup differences were assessed based on the data from the treatment groups at the time of dosing. T-tests were used to compare treatment groups with the vehicle control group, with p < 0.05 considered significant. All data were analyzed using SPSS 17.0.

[0661] The experimental results are as follows:

[0662] (1) Animal Observation

[0663] During the entire experimental period, the animals were observed once a day, and no obvious abnormal reactions were observed in the appearance, behavioral activities, glandular secretions, and respiration of all animals.

[0664] (2) Tumor volume and experimental indicators

[0665] Tumor volume was measured, and tumor proliferation rate and tumor growth inhibition rate were calculated in CB17 SCID mice bearing subcutaneous xenografts of human acute myeloid leukemia cell lines MV4-11 after treatment with vehicle and test compounds. The effects of the test compounds on subcutaneous xenografts of human acute myeloid leukemia cell lines MV4-11 were analyzed.

[0666] (3) Tumor growth

[0667] There was no significant upward trend in tumor volume during the administration of the test molecule group, as shown in Table 24.

[0668] Table 24: Tumor volume of each group at different time points Note: Mean ± SEM. *. Tumor volumes in the treatment group and the vehicle control group were analyzed by T-test at different time points. * indicates p < 0.05, and none indicates p > 0.05.

[0669] Tumor proliferation rate: See Table 25 for details.

[0670] Table 25: Effects of the test drugs on tumor proliferation rate in the MV4-11 human acute myeloid leukemia cell xenograft model Note: a. Days after grouping; b. Tumor proliferation rate T / C (%) = TRTV / CRTV × 100;

[0671] Tumor growth inhibition rate: See Table 26.

[0672] Table 26: Tumor growth inhibition rate of the test drugs in the MV4-11 human acute myeloid leukemia cell xenograft model Note: a. Days after grouping; b. Tumor growth inhibition rate TGI (%) = (1-T RTV / C RTV )×100;

[0673] Compared with the data of PG-D21 (last administration), the tumor volume of the tumor-bearing mice in the vehicle control group reached 1460 mm 3 Compared with the vehicle control group, the test drug group tested molecule, 10 mg / kg, BIW (tumor volume = 374 mm 3 , TGI=78.24%, p<0.05) showed a significant tumor inhibitory effect.

[0674] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. An anti-CD33 single-domain antibody, characterized in that: 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: 9-11; CDR2 shown in any one of SEQ ID NO: 12 and SEQ ID NO: 13; CDR3 shown in any one of SEQ ID NOs: 14-16; The 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.

2. The single domain antibody 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: 9, CDR2 shown in SEQ ID NO: 12, and CDR3 shown in SEQ ID NO: 14; (2) CDR1 shown in SEQ ID NO: 10, CDR2 shown in SEQ ID NO: 13, and CDR3 shown in SEQ ID NO: 15; (3) CDR1 shown in SEQ ID NO: 11, CDR2 shown in SEQ ID NO: 13, and CDR3 shown in SEQ ID NO: 16; (4) CDR1 shown in SEQ ID NO: 11, CDR2 shown in SEQ ID NO: 13, and CDR3 shown in SEQ ID NO:

16.

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

4. The single domain antibody according to claim 3, characterized in that: The heavy chain further includes a framework region FR; the framework region FR includes 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 any one of SEQ ID NOs: 17-18; FR2 shown in any one of SEQ ID NOs: 19-21; FR3 shown in any one of SEQ ID NOs: 22-24; FR4 shown in SEQ ID NO:25-26; The 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.

5. The single domain antibody according to claim 1, wherein: The amino acid sequence of the single-domain antibody is selected from any one of SEQ ID NOs: 1-4, or has at least 80% sequence homology with any one of SEQ ID NOs: 1-4. The humanized antibody or Fc fusion antibody of the single-domain antibody according to any one of claims 1 to 5.

7. An anti-CD123 single-domain antibody, characterized in that: 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:41-46; CDR2 shown in any one of SEQ ID NOs:47-51; CDR3 shown in any one of SEQ ID NOs: 52-57; The amino acid sequence includes derivative sequences formed by optionally adding, deleting, modifying and / or replacing 1-5 amino acids and capable of retaining CD123 binding affinity.

8. The single domain antibody according to claim 7, characterized in that: The CDR1, CDR2 and CDR3 are selected from the following groups: (1) CDR1 shown in SEQ ID NO:41, CDR2 shown in SEQ ID NO:47, and CDR3 shown in SEQ ID NO:52; (2) CDR1 shown in SEQ ID NO:42, CDR2 shown in SEQ ID NO:47, and CDR3 shown in SEQ ID NO:52; (3) CDR1 shown in SEQ ID NO:43, CDR2 shown in SEQ ID NO:48, and CDR3 shown in SEQ ID NO:53; (4) CDR1 shown in SEQ ID NO:44, CDR2 shown in SEQ ID NO:49, and CDR3 shown in SEQ ID NO:54; (5) CDR1 shown in SEQ ID NO:45, CDR2 shown in SEQ ID NO:50, and CDR3 shown in SEQ ID NO:55; (6) CDR1 shown in SEQ ID NO:46, CDR2 shown in SEQ ID NO:51, and CDR3 shown in SEQ ID NO:56; (7) CDR1 shown in SEQ ID NO:46, CDR2 shown in SEQ ID NO:51, and CDR3 shown in SEQ ID NO:

57.

9. The single domain antibody according to claim 7, characterized in that: The single domain antibody is composed of heavy chains.

10. The single domain antibody according to claim 9, characterized in that: The heavy chain further includes a framework region FR, which includes the amino acid sequences of FR1, FR2, FR3 and FR4, and the framework region FR is selected from the following amino acid sequences: FR1 or a variant of FR1 shown in any one of SEQ ID NOs: 17-18, 58; FR2 or a variant of FR2 shown in any one of SEQ ID NOs: 59-64; FR3 or a variant of FR3 shown in any one of SEQ ID NOs: 65-71; FR4 or a variant of FR4 shown in any one of SEQ ID NOs: 25-26; The amino acid sequence includes derivative sequences formed by optionally adding, deleting, modifying and / or replacing 1-5 amino acids and capable of retaining CD123 binding affinity.

11. The single domain antibody according to claim 7, characterized in that: The amino acid sequence of the single-domain antibody is selected from any one of SEQ ID NOs: 27-33, or has at least 80% sequence homology with any one of SEQ ID NOs: 27-33. 12 . The humanized antibody or Fc fusion antibody of the single-domain antibody according to claim 7 .

13. A bispecific polypeptide, characterized in that: The polypeptide comprises a first binding portion that specifically binds to CD33 and a second binding portion that specifically binds to CD123, wherein the polypeptide comprises or consists of at least two immunoglobulin single variable domains, the variable domains comprising three complementarity determining regions CDR1, CDR2 and CDR3, and the complementarity determining regions comprising the following amino acid sequence: The CDR1 of the first binding moiety comprises the amino acid sequence of SEQ ID NO: 11, the CDR2 comprises the amino acid sequence of SEQ ID NO: 13, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 16; the CDR1 of the second binding moiety comprises the amino acid sequence of SEQ ID NO:46, the CDR2 comprises the amino acid sequence of SEQ ID NO:51, and the CDR3 comprises the amino acid sequence of SEQ ID NO:57; The amino acid sequence includes derivative sequences formed by optionally adding, deleting, modifying and / or replacing 1-3 amino acids, and capable of retaining the binding affinity to CD123 and CD33, respectively.

14. The polypeptide according to claim 13, characterized in that: The variable domain is a variable domain of a heavy chain antibody, wherein the variable domain of the first binding portion comprises the amino acid sequence of SEQ ID NO: 4 or has at least 90% identity with the amino acid sequence of SEQ ID NO: 4; the variable domain of the second binding portion comprises the amino acid sequence of SEQ ID NO: 33 or has at least 90% identity with the amino acid sequence of SEQ ID NO:

33.

15. The polypeptide according to claim 14, characterized in that: The variable domain of the first binding moiety consists of the amino acid sequence of SEQ ID NO: 4, and the variable domain of the second binding moiety consists of the amino acid sequence of SEQ ID NO:

33.

16. A bispecific antibody, characterized in that: The antibodies comprise a first full-length antibody and a second full-length antibody, wherein the first full-length antibody comprises a heavy chain variable domain and an Fc region polypeptide that specifically binds to a first binding portion of CD33, and the second full-length antibody comprises a heavy chain variable domain and an Fc region polypeptide that specifically binds to a second binding portion of CD123, wherein the heavy chain variable domain comprises three complementarity determining regions (CDR1, CDR2, and CDR3), and the CDR1 of the first full-length antibody comprises the amino acid sequence of SEQ ID NO: 11, the CDR2 comprises the amino acid sequence of SEQ ID NO: 13, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 16; the CDR1 of the second full-length antibody comprises the amino acid sequence of SEQ ID NO: 46, the CDR2 comprises the amino acid sequence of SEQ ID NO: 51, and the CDR3 comprises the amino acid sequence of SEQ ID NO: 57; The amino acid sequence includes derivative sequences formed by optionally adding, deleting, modifying and / or replacing 1-3 amino acids, and capable of retaining the binding affinity to CD123 and CD33, respectively.

17. The bispecific antibody according to claim 16, characterized in that: The Fc region polypeptide is derived from a human IgG class Fc region polypeptide, and comprises a modified first variant Fc region polypeptide and a modified second variant Fc region polypeptide; wherein the carboxyl terminus of the heavy chain variable domain of the first binding moiety is coupled to the amino terminus of the modified first variant Fc region polypeptide, and the carboxyl terminus of the heavy chain variable domain of the second binding moiety is coupled to the amino terminus of the modified second variant Fc region polypeptide.

18. The bispecific antibody according to claim 16, characterized in that: The variable domain of the first binding moiety consists of the amino acid sequence of SEQ ID NO: 4, and the variable domain of the second binding moiety consists of the amino acid sequence of SEQ ID NO:

33.

19. The bispecific antibody according to claim 16, wherein: The first full-length antibody consists of the amino acid sequence of SEQ ID NO: 74, and the second full-length antibody consists of the amino acid sequence of SEQ ID NO:

75.

20. An isolated nucleotide molecule, characterized in that: The nucleic acid encodes the polypeptide of any one of claims 13 to 15, or the antibody of any one of claims 16 to 19.

21. The nucleotide molecule according to claim 20, characterized in that: The nucleotide sequence encoding the first full-length antibody is shown in SEQ ID NO: 76, and the nucleotide sequence encoding the second full-length antibody is shown in SEQ ID NO:

77.

22. A recombinant protein, characterized in that: The recombinant protein comprises the antibody of any one of claims 1 to 12 and 16 to 19.

23. An isolated nucleic acid, characterized in that: The nucleic acid encodes the antibody of any one of claims 1 to 12 and 16 to 19, or the recombinant protein of claim 22.

24. An expression vector, characterized in that: It comprises a nucleotide molecule encoding the antibody according to any one of claims 1 to 12 and 16 to 19 or the nucleic acid according to claim 23 .

25. A host cell, characterized in that: It expresses the antibody of any one of claims 1 to 12 and 16 to 19, or comprises the nucleic acid of claim 23, or comprises the expression vector of claim 24; Optionally, the host cell is selected from a prokaryotic cell, a eukaryotic cell or a bacteriophage.

26. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises an antibody selected from any one of claims 1 to 12 and 16 to 19, a recombinant protein of claim 22, a nucleic acid of claim 23, an expression vector of claim 24, a host cell of claim 25, or a combination thereof; and a pharmaceutically acceptable carrier.

27. Use of the antibody according to any one of claims 1 to 12 and 16 to 19 or the pharmaceutical composition according to claim 26 in the preparation of a medicament for treating a disease.

28. The use according to claim 27, characterized in that The disease is a tumor.

29. The method according to claim 27, wherein: The disease is a blood tumor disease.

30. The method according to claim 27, wherein: The disease is acute myeloid leukemia.

31. A method for producing an antibody, comprising: (a) cultivating the host cell of claim 25 and (b) recovering the antibody.

32. An immunoconjugate, characterized in that: Comprising the polypeptide according to any one of claims 13 to 15 or the antibody according to any one of claims 16 to 19, or a combination thereof.

33. The immunoconjugate according to claim 32, characterized in that: The conjugates include cytotoxins, biologically active proteins, or radioactive isotopes.

34. An antibody-drug conjugate, characterized in that: Comprising the polypeptide according to any one of claims 13 to 15 or the antibody according to any one of claims 16 to 19, or a combination thereof.

35. A method for treating tumor diseases using the antibody of any one of claims 1 to 12 and 16 to 19, or the pharmaceutical composition of claim 26, the immunoconjugate of claims 32 to 33, and the antibody-drug conjugate of claim 34.

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