Antigen binding protein bound with CD123 and application thereof

Abstract

The invention belongs to the field of immunology, and relates to an antigen binding protein combined with CD123 and application of the antigen binding protein. The antigen binding protein bound with CD123 comprises an anti-CD123 single-domain antibody, the anti-CD123 single-domain antibody is composed of heavy chains, and the heavy chains comprise a heavy chain CDR1 as shown in any one of SEQ ID NO: 25 to SEQ ID NO: 30, a heavy chain CDR2 as shown in SEQ ID NO: 31 and a heavy chain CDR3 as shown in any one of SEQ ID NO: 32 to SEQ ID NO: 33. The CD123 single-domain antibody has the beneficial effects that the specific CD123 single-domain antibody is screened by using a biological gene engineering technology, and the affinity of the antibody is relatively good.

Description

Technical Field

[0001] The present invention relates to amino acid sequences directed against interleukin-3 receptor (IL-3R) that can specifically bind (as defined herein), as well as compounds or constructs, and in particular proteins and polypeptides, comprising or essentially consisting of one or more of said amino acid sequences. Background Art

[0002] Interleukin-3 (IL-3) is one of the main components of HGFs produced by hematological tumor cells. It can induce proliferation, anti-apoptosis and differentiation signals after binding to its cell surface receptor IL-3R. CD123 is also known as IL-3Ra, which is the α chain of IL-3R. After IL-3Ra binds to IL-3 and recruits the IL-3Rβ chain, the complex formed can cascade activate the downstream signal JAK2 in the cell, mediating cell proliferation and survival.

[0003] CD123 has been reported to be overexpressed on malignant cells in a wide range of hematological malignancies, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) (et al., Haematologica 86 (12): 1261-1269, 2001). Overexpression of CD123 is associated with poor prognosis in AML (Tettamanti et al., Br. J. Haematol. 161: 389-401, 2013). AML and MDS are believed to arise in and be immortalized by a small population of leukemic stem cells (LSCs) that are usually dormant (i.e., non-rapidly dividing cells), and are therefore resistant to cell death (apoptosis) and conventional chemotherapeutic agents. LSCs are characterized by overexpression of CD123, which is absent in the corresponding normal hematopoietic stem cell population in normal human bone marrow (Jin et al., Blood 113:6603-6610, 2009; Jordan et al., Leukemia 14:1777-1784, 2000). CD123 expression is also associated with a variety of other malignancies / premalignancies: chronic myeloid leukemia (CML) progenitor cells (including primitive cell crisis CML); Hodgkin's Reed-Sternberg (RS) cells; transformed non-Hodgkin's lymphoma (NHL); some chronic lymphocytic leukemias (CLL) (CD11c+); subsets of acute T lymphoblastic leukemia (T-ALL) (16%, the most immature, mainly adults), plasmacytoid dendritic cells (pDC) (DC2) malignancies, and CD34+ / CD38- myelodysplastic syndrome (MDS) myeloid cell malignancies.

[0004] Currently, drugs targeting CD123 include ADC drugs, bispecific antibodies, and CAR-T drugs. For example, IMGN632 is an ADC drug targeting CD123, which was granted Breakthrough Therapy Designation (BTD) by the U.S. 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 drug developed by Janssen, a subsidiary of Johnson & Johnson. Summary of the invention

[0005] The object of the present invention is to provide an antigen binding protein capable of specifically binding to CD123 and uses thereof.

[0006] The first aspect of the present invention provides an antigen binding protein that binds to CD123, wherein the antigen binding protein comprises a single domain antibody against CD123, wherein the single domain antibody is composed of a heavy chain, and the heavy chain comprises

[0007] a) a heavy chain CDR1 or a variant of CDR1 as shown in any one of SEQ ID NO:25 to SEQ ID NO:30, wherein the variant of CDR1 comprises a substitution of at most 2 amino acids in the CDR1;

[0008] b) the heavy chain CDR2 or a variant of CDR2 as shown in SEQ ID NO: 31; the variant of CDR2 comprises a substitution of up to 2 amino acids in the CDR2;

[0009] c) a heavy chain CDR3 or a variant of CDR3 as shown in any one of SEQ ID NO:32 to SEQ ID NO:33, wherein the variant of CDR3 comprises a substitution of at most 2 amino acids in the CDR3.

[0010] Preferably, the amino acid sequence of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 is one of the following (1)-(7):

[0011] (1) CDR1 shown in SEQ ID NO:25, CDR2 shown in SEQ ID NO:31, and CDR3 shown in SEQ ID NO:32;

[0012] (2) CDR1 shown in SEQ ID NO:25, CDR2 shown in SEQ ID NO:31, and CDR3 shown in SEQ ID NO:33;

[0013] (3) CDR1 shown in SEQ ID NO:26, CDR2 shown in SEQ ID NO:31, and CDR3 shown in SEQ ID NO:32;

[0014] (4) CDR1 shown in SEQ ID NO:27, CDR2 shown in SEQ ID NO:31, and CDR3 shown in SEQ ID NO:32;

[0015] (5) CDR1 shown in SEQ ID NO:28, CDR2 shown in SEQ ID NO:31, and CDR3 shown in SEQ ID NO:32;

[0016] (6) CDR1 shown in SEQ ID NO:29, CDR2 shown in SEQ ID NO:31, and CDR3 shown in SEQ ID NO:32;

[0017] (7) CDR1 shown in SEQ ID NO:30, CDR2 shown in SEQ ID NO:31, and CDR3 shown in SEQ ID NO:32.

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

[0019] In one embodiment, one to five arbitrary amino acid residues in any one or more CDRs of the heavy chain CDR1, CDR2 and CDR3 can be replaced by their conservative amino acids, respectively. Specifically, in the heavy chain CDR1, 1 to 5 amino acid residues can be replaced by their conservative amino acids; in the heavy chain CDR2, 1 to 5 amino acid residues can be replaced by their conservative amino acids; in the heavy chain CDR3, 1 to 5 amino acid residues can be replaced by their conservative amino acids.

[0020] As used herein, the term "sequence homology" refers to the extent to which two (nucleotide or amino acid) sequences have identical residues at the same position in an alignment, and is usually expressed as a percentage. Preferably, homology is determined over the entire length of the sequences being compared. Thus, two copies having exactly the same sequence have 100% homology.

[0021] In some embodiments, a sequence that replaces only one or a few amino acids compared to the aforementioned sequence, for example, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions, can also achieve the purpose of the invention. These variant forms include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and optimally 1-10) amino acid deletions, insertions and / or substitutions, and addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. In fact, when determining the degree of sequence homology between two amino acid sequences or when determining the combination of CDR1, CDR2 and CDR3 in a single domain antibody, a technician may consider so-called "conservative" amino acid substitutions, and in the case of substitutions, the substitutions will preferably be conservative amino acid substitutions. The conservative amino acids, which can generally be described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue having a similar chemical structure, and the substitution has little or substantially no effect on the function, activity or other biological properties of the polypeptide. The conservative amino acid substitutions are common in the art. For example, conservative amino acid substitutions are substitutions in which one or a few amino acids in the following groups (a)-(d) are replaced by another or a few amino acids in the same group: (a) polar negatively charged residues and their uncharged amides: Asp, Asn, Glu, Gln; (b) polar positively charged residues: His, Arg, Lys; (c) aromatic residues: Phe, Trp, Tyr; (d) aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Gly, Pro, Met, Leu, Ile, Val, Cys. Particularly preferred conservative amino acid substitutions are as follows: Asp is replaced by Glu; Asn is replaced by Gln or His; Glu is replaced by Asp; Gln is replaced by Asn; His is replaced by Asn or Gln; Arg is replaced by Lys; Lys is replaced by Arg, Gln; Phe is replaced by Met, Leu, Tyr; Trp is replaced by Tyr; Tyr is replaced by Phe, Trp; Ala is replaced by Gly or Ser; Ser is replaced by Thr; Thr is replaced by Ser; Gly is replaced by Ala or Pro; Met is replaced by Leu, Tyr or Ile; Leu is replaced by Ile or Val; Ile is replaced by Leu or Val; Val is replaced by Ile or Leu; Cys is replaced by Ser. In addition, those skilled in the art know that the framework region sequences FR1-4 are not immutable, and the sequences of FR1-4 can adopt conservative sequence variants of the sequences disclosed in the present invention.

[0022] In the present invention, the antigen binding protein bound to CD123 is a single domain antibody against CD123. The meaning of "single domain antibody against CD123" includes not only complete single domain antibodies, but also fragments, derivatives and analogs of the single domain antibody 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 of 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 substitution group in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound (such as a compound that prolongs the half-life of a polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusion of an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with an Fc tag). Based on the teachings herein, these fragments, derivatives and analogs are well within the scope known to those skilled in the art.

[0023] 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 amino acid sequences of the framework region FR are respectively:

[0024] FR1 or a variant of FR1 as shown in any one of SEQ ID NOs: 17-18, wherein the variant of FR1 comprises a substitution of up to 5 amino acids in FR1;

[0025] FR2 or a variant of FR2 as shown in SEQ ID NO: 19, wherein the variant of FR2 comprises a substitution of up to 5 amino acids in FR2;

[0026] FR3 or a variant of FR3 as shown in any one of SEQ ID NOs: 20-23, wherein the variant of FR3 comprises a substitution of up to 5 amino acids in FR3;

[0027] FR4 or a variant of FR4 as shown in SEQ ID NO:24, wherein the variant of FR4 comprises a substitution of up to 5 amino acids in the FR4.

[0028] The second aspect of the present invention is to provide an amino acid sequence of an antigen binding protein that can bind to CD123, wherein the antigen binding protein that binds to CD123 is a single domain antibody against CD123, and the amino acid sequences of the single domain antibodies are shown in SEQ ID NOs: 1-8, respectively, or the single domain antibodies have at least 80% sequence homology with the amino acid sequences of SEQ ID NOs: 1-8, and can specifically bind to the CD123 protein.

[0029] 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 with an amino acid sequence selected from SEQ ID NO: 1-8 or SEQ ID NO: 1-8, and is capable of specifically binding to CD123 protein.

[0030] The third aspect of the present invention is to provide any of the aforementioned Fc fusion proteins consisting of an antigen binding protein that binds to CD123 and Fc, or a humanized antibody of the aforementioned antigen binding protein that binds to CD123.

[0031] A fourth aspect of the present invention is to provide a nucleotide molecule encoding the aforementioned antigen binding protein that binds to CD123 or the aforementioned Fc fusion protein or the aforementioned humanized antibody, whose nucleotide sequence is shown in SEQ ID NO: 9-16, respectively, or has at least 95% sequence homology with any one of SEQ ID NO: 9-16.

[0032] In one embodiment, the nucleic acid molecule encoding the antigen binding protein bound to CD123 is selected from SEQ ID NO: 9-16 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology with a nucleotide sequence selected from SEQ ID NO: 9-16, and the antigen binding protein bound to CD123 encoded by it can specifically bind to anti-CD123 protein.

[0033] A fifth aspect of the present invention is to provide an expression vector comprising a nucleotide molecule encoding an antigen binding protein or Fc fusion protein or a humanized antibody that binds to CD123, wherein the nucleotide sequence is as shown in SEQ ID NO: 9-16 or has at least 80% sequence homology with any one of SEQ ID NO: 9-16.

[0034] 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 protein or humanized antibody is integrated into RJK-V4-hFC1 by genetic engineering means), and other general expression vectors can also be selected as needed.

[0035] The sixth aspect of the present invention is to provide a host cell capable of expressing the aforementioned antigen binding protein, Fc fusion protein or humanized antibody bound to CD123, or an expression vector comprising the aforementioned host cell. Preferably, the host cell is a bacterial cell, a fungal cell or a mammalian cell.

[0036] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell, including bacteria and fungi.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] In another preferred embodiment, the host cell is a suspension ExpiCHO-S cell.

[0042] In another preferred embodiment, the host cell is a suspension 293F cell.

[0043] The seventh aspect of the present invention is to provide a recombinant protein comprising the aforementioned antigen binding protein that binds to CD123. The recombinant protein may be a single domain antibody as shown in the aforementioned SEQ ID NO: 1-8, or a single domain antibody having at least 80% homology with SEQ ID NO: 1-8, or a multi-epitope antibody, a multi-specific antibody, and a multivalent antibody; for example, the multi-epitope antibody may be composed of more than one sequence in SEQ ID NO: 1-8; the multivalent antibody may be composed of one of the sequences in SEQ ID NO: 1-8 repeated several times; the multi-specific antibody includes but is not limited to a bispecific antibody, and a tri-specific antibody; in addition, the recombinant protein may be a fragment, derivative, and analog of the aforementioned antibody.

[0044] An eighth aspect of the present invention is to provide a pharmaceutical composition comprising the aforementioned antigen binding proteins bound to CD123 and a pharmaceutically acceptable carrier. Typically, these substances can be formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically 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 differs by about 2 from the isoelectric point of the antibody).

[0045] The pharmaceutical composition of the present invention can be directly used to bind to anti-CD123 protein molecules.

[0046] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the aforementioned 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 match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions.

[0047] The ninth aspect of the present invention is to provide a medicament for treating a disease, comprising the aforementioned antigen binding protein that binds to CD123 as an active ingredient.

[0048] The tenth aspect of the present invention is to provide a kit for detecting CD123 levels, which contains the aforementioned antigen binding protein that binds to CD123. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, a buffer, and the like.

[0049] 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 markers, detection substrates, etc. The detection kit can be an in vitro diagnostic device.

[0050] In a preferred embodiment, the kit further contains a secondary antibody and an enzyme or fluorescent or radioactive label for detection, and a buffer.

[0051] In a preferred embodiment, the second antibody of the kit may be the aforementioned single domain antibody against CD123 (as an anti-antibody), and may be a single domain antibody, a monoclonal antibody, a polyclonal antibody or any other form of an antibody.

[0052] In an eleventh aspect of the present invention, a method for producing an antigen binding protein (anti-CD123 single domain antibody) that binds to CD123 is provided, comprising the steps of:

[0053] (a) culturing the host cell according to the sixth aspect of the present invention under conditions suitable for producing the single domain antibody, thereby

[0054] Obtaining a culture containing the anti-CD123 single domain antibody; and

[0055] (b) isolating or recovering the anti-CD123 single domain antibody from the culture; and

[0056] (c) optionally, purifying and / or modifying the anti-CD123 single domain antibody obtained in step (b).

[0057] The twelfth aspect of the present invention is to provide the use of the aforementioned antigen binding protein that binds to CD123 or the aforementioned pharmaceutical composition in the preparation of a drug for treating a disease.

[0058] The thirteenth aspect of the present invention is to provide a method for treating a disease using the aforementioned antigen binding protein that binds to CD123 (anti-CD123 single domain antibody) or the aforementioned pharmaceutical composition.

[0059] Beneficial Effects

[0060] Compared with the prior art, the beneficial effects of the present invention are:

[0061] (1) The single-domain antibody of the present invention is specific for anti-CD123 protein with a correct spatial structure.

[0062] (2) The single-domain antibody obtained in the present invention has a flexible expression system selection, and can be expressed in a prokaryotic system or a eukaryotic system of yeast cells or mammalian cells. In addition, the expression cost in the prokaryotic expression system is low, which can reduce the later production cost.

[0063] (3) The single-domain antibodies obtained by the present invention are easy to modify in multiple combinations. Multivalent and multispecific antibodies can be obtained by simple concatenation through genetic engineering. In addition, their immune heterogeneity is very low and they will not produce a strong immune response without humanization.

[0064] (4) The single domain antibody obtained by the present invention has a wider affinity range. Before affinity maturation, its affinity range can range from nM level to pM level, providing multiple options for antibodies with different uses in the later stage. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] In order to more clearly illustrate the technical solution of the present application, the drawings required for use in the embodiments are briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0066] Figure 1 The antibody-antigen binding dose-effect curve measurement profile (Tab1, Tab4, hIgG, 1A3) in Example 12;

[0067] Figure 2 The antibody-antigen binding dose-effect curve measurement graph (1A6) in Example 12;

[0068] Figure 3 The antibody-antigen binding dose-effect curve measurement profile (1C10, 1D1) in Example 12;

[0069] Figure 4 The antibody-antigen binding dose-effect curve measurement profile (1D4, 1F6, 1G7) in Example 12;

[0070] Figure 5 The antibody-antigen binding dose-effect curve measurement spectrum (2A9) in Example 12;

[0071] Figure 6 The ADCC effect determination profile of the antibodies in Example 13 (Tab4, hIgG, 1A6);

[0072] Figure 7 The ADCC effect measurement spectrum (1F6) of the antibody in Example 13;

[0073] Figure 8 The ADCC effect measurement spectrum (2A9) of the antibody in Example 13;

[0074] Fig. 9 The ADCC effect measurement spectrum (1A3) of the antibody in Example 13;

[0075] Fig.10 The ADCC effect determination profiles of the antibodies in Example 13 (1C10, 1D1);

[0076] Fig.11 The ADCC effect measurement profiles of the antibodies in Example 13 (1D4, 1G7). DETAILED DESCRIPTION

[0077] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0078] As used herein, "single domain antibodies" (sdAb, also referred to as nanobodies or VHHs by developer Ablynx) are well known to those skilled in the art. Single domain antibodies are antibodies whose complementary determining regions are part of a single domain polypeptide. Thus, single domain antibodies comprise a single complementary determining region (a single CDR1, a single CDR2, and a single CDR3). Examples of single domain antibodies are antibodies with only heavy chains (which naturally do not comprise light chains), single domain antibodies derived from conventional antibodies, and engineered antibodies.

[0079] Single domain antibodies can be derived from any species, including mice, humans, camels, llamas, goats, rabbits and cattle. For example, naturally occurring VHH molecules can be derived from antibodies provided by camelid species (e.g., camels, dromedaries, llamas and guanacos). Like complete antibodies, single domain antibodies are able to selectively bind to specific antigens. Single domain antibodies can contain only the variable domains of immunoglobulin chains, which have CDR1, CDR2 and CDR3 and framework regions.

[0080] As used herein, the term "Fc fusion protein" refers to a novel protein produced by fusing the Fc segment of a target antibody with a functional protein molecule having biological activity using genetic engineering technology.

[0081] The term "humanized antibody" refers to an antibody obtained by fusing the heavy chain variable region of a target antibody (such as an animal antibody) with the constant region of a human antibody, or an antibody obtained by transplanting the complementary determining region (CDR1-3 sequence) of a target antibody into the variable region of a human antibody, or an antibody obtained by mutating the amino acids of the target antibody according to the characteristics of the human antibody framework region (FR1-4). Humanized antibodies can be synthesized or by site-directed mutagenesis.

[0082] 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.

[0083] This patent is to prepare the target protein and the truncated form of the target protein through genetic engineering technology, and then immunize the Alxa Bactrian camel in Inner Mongolia with the obtained antigen protein. After multiple immunizations, the peripheral blood lymphocytes or spleen cells of the camel are obtained. The variable region coding sequence of the camel-derived antibody is recombined into the phage display vector through genetic engineering. The specific antibodies against the antigen protein are screened through phage display technology, and their ability to bind to the antigen and their application in inhibiting tumors, autoimmunity and inflammatory diseases are further tested.

[0084] The above technical solution is now broken down and explained in detail in the form of specific embodiments:

[0085] Example 1: Preparation of human CD123 protein:

[0086] The human recombinant extracellular domain protein used in this patent is obtained by the company's own expression and purification. The expression vector design scheme of the human recombinant CD123 protein is as follows:

[0087] (1) The coding sequence of CD123 was retrieved from NCBI and its accession number is NM_006023.2. The accession number of the amino acid sequence encoded by this sequence is NP_006014.2.

[0088] (2) The nucleotide sequence encoding CD123 protein (amino acids 19 to 300) was cloned into a vector (e.g., pcDNA3.4) by gene synthesis. The constructed vector was subjected to Sanger sequencing, and the original sequence was compared. After confirmation, the recombinant plasmid was extracted in large quantities, endotoxins were removed, and suspended 293F cells were transfected for target protein expression and purification, with a purity of more than 90%, meeting animal immunization requirements.

[0089] Example 2: Construction of a single domain antibody library against CD123 protein:

[0090] 1 mg of the human recombinant CD123 protein purified in Example 1 was mixed with an equal volume of Freund's complete adjuvant to immunize an Alxa Bactrian camel in Inner Mongolia, once a week for a total of 7 consecutive immunizations. Except for the first immunization, the remaining six immunizations were performed by mixing 1 mg of CD123 protein with an equal volume of Freund's incomplete adjuvant for animal immunization. The immunization process was to centrally stimulate the camel to produce antibodies against the CD123 protein.

[0091] After the animal immunization, 150 mL of peripheral blood lymphocytes were extracted from the camel, and RNA was extracted from the cells. The extracted total RNA was used to synthesize cDNA, and VHH (antibody heavy chain variable region) was amplified using cDNA as a template through a nested PCR reaction.

[0092] Then, the pMECS vector and VHH fragment were digested with restriction endonucleases, and the digested fragments and vector were linked. The linked fragments were electroporated into competent cells TG1 to construct a phage display library of CD123 protein and measure the library capacity. The library capacity was about 1×10 9 At the same time, the correct insertion rate of the library in the target fragment was detected by colony PCR identification.

[0093] 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%.

[0094] Example 3: Screening of single domain antibodies against CD123 protein:

[0095] 200 μL of the recombinant TG1 cells in Example 2 were cultured in 2×TY medium, during which 40 μL of helper phage VCSM13 was added to infect the TG1 cells and cultured overnight to amplify the phages. The next day, the phages were precipitated with PEG / NaCl and the amplified phages were collected by centrifugation.

[0096] 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 set up at the same time. On the second day, 200 μL of 3% skim milk was added and the plate was blocked at room temperature for 2 h. After blocking, 100 μL of amplified phage library (approximately 2 × 10 11 After 1 hour of incubation, the cells were washed 15 times with PBS + 0.05% Tween-20 to remove unbound phages.

[0097] The phages specifically bound to the CD123 protein were dissociated with trypsin at a final concentration of 25 mg / mL and infected with Escherichia coli TG1 cells in the logarithmic growth phase. The cells were cultured at 37°C for 1 h to produce and collect phages for the next round of screening. The same screening process was repeated for one round to gradually obtain enrichment.

[0098] Example 4: Screening of specific positive clones for CD123 using phage ELISA:

[0099] According to the screening method in Example 3 above, 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 the screening and inoculated into 96 deep-well plates in 2×TY medium containing 100 μg / mL ampicillin, and a blank control was set. After culturing at 37°C to the logarithmic phase, IPTG with a final concentration of 1 mM was added and cultured overnight at 28°C.

[0100] The crude antibody was obtained by osmotic swelling method; CD123 recombinant protein was released into 100mM NaHCO3 at pH8.3 and 100μg protein was coated in ELISA plate at 4℃ overnight. 100μL of the crude antibody extract was transferred to the ELISA plate with antigen added and incubated at room temperature for 1h; unbound antibody was washed with PBST, and 100μl of Mouse Anti-HAtag Antibody (HRP) (mouse anti-HA horseradish peroxidase labeled antibody, Thermo Fisher) diluted 1:2000 was added and incubated at room temperature for 1h; unbound antibody was washed with PBST, horseradish peroxidase colorimetric solution was added, and after reacting at 37℃ for 15min, stop solution was added and the absorbance value was read at 450nm wavelength on the microplate reader.

[0101] When the OD value of the sample well is more than 5 times greater than that of the control well, it is determined to be a positive clone well; the bacteria in the positive clone well are transferred and shaken in LB medium containing 100 μg / mL ampicillin to extract the plasmid and perform sequencing.

[0102] The gene sequences of each clone were analyzed using the sequence alignment software VectorNTI, and strains with the same CDR1, CDR2 and CDR3 sequences were considered to be the same clone, while strains with different sequences were considered to be different clones, and single-domain antibodies specific to the CD123 protein were finally obtained (including single-domain antibodies 1A6, 1F6, 2A9, 1A3, 1C10, 1D4, 1G7, 1D1).

[0103] The amino acid sequence of the antibody is a FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 structure, 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.

[0104] The amino acid sequences of the single domain antibodies 1A3, 1A6, 1C10, 1D1, 1D4, 1F6, 1G7, and 2A9 are shown in SEQ ID NOs: 1-8, and their nucleotide sequences are shown in SEQ ID NOs: 9-16.

[0105] The CDR sequences of the eight single-domain antibodies are shown in Tables 1 to 3. The FR sequences of the eight single-domain antibodies are shown in Tables 4 to 7.

[0106] Table 1 CDR1 sequences and numbers of 8 single domain antibodies

[0107]

[0108] Table 2 CDR2 sequences and numbers of 8 single domain antibodies

[0109]

[0110] Table 3 CDR3 sequences and numbers of 8 single domain antibodies

[0111]

[0112] Table 4 FR1 sequences and numbers of 8 single domain antibodies

[0113]

[0114] Table 5 FR2 sequences and numbers of 8 single domain antibodies

[0115]

[0116] Table 6 FR3 sequences and numbers of 8 single domain antibodies

[0117]

[0118] Table 7 FR4 sequences and numbers of 8 single domain antibodies

[0119]

[0120] Table 8 Amino acid sequences of 8 single domain antibodies

[0121]

[0122]

[0123] Table 9 Amino acid sequences of 8 single domain antibodies

[0124]

[0125]

[0126] Example 5: Purification and expression of specific single domain antibodies against CD123 protein in host bacteria Escherichia coli

[0127] The plasmids (pMECS-VHH) of different clones obtained by sequencing analysis in Example 4 were electrotransformed into Escherichia coli HB2151, and spread on LB+amp+glucose culture plates containing ampicillin and glucose, and cultured at 37°C overnight; a single colony was selected and inoculated into 5 mL of LB culture medium containing ampicillin, and cultured at 37°C in a shaking incubator overnight.

[0128] Inoculate 1 mL of overnight cultured bacteria into 330 mL of TB culture medium, culture at 37°C in a shaking incubator, add 1 M IPTG when the OD600nm value reaches 0.6-0.9, and culture overnight in a shaking incubator at 28°C; centrifuge to collect E. coli, and use the osmotic swelling method to obtain a crude antibody extract;

[0129] The antibody was purified by nickel column affinity chromatography.

[0130] Example 6: Construction of Fc fusion antibody eukaryotic expression vector of anti-CD123 single domain antibody

[0131] (1) Subcloning the target sequence obtained in Example 4 into a eukaryotic expression vector: The antibody screened in Example 4 was sequenced by Sanger sequencing to obtain its nucleotide sequence;

[0132] (2) The above nucleotide sequences (SEQ ID NOs: 9-16 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;

[0133] (3) transforming the recombinant eukaryotic expression vector constructed in step (2) into DH5α Escherichia coli, culturing for plasmid extraction, and removing endotoxin;

[0134] (4) Sequencing the extracted plasmid for further identification;

[0135] (5) The confirmed recombinant vector is prepared for subsequent eukaryotic cell transfection and expression, and the Fc protein of VHH is expressed by the method of Example 7 or 8, and the above antibody is purified by the method of Example 9.

[0136] Example 7: Expression of single domain antibodies against CD123 protein in suspension ExpiCHO-S cells

[0137] (1) 3 days before transfection, 2.5×10 5 / mL cell passaging and expansion of ExpiCHO-S TM Transfer the calculated volume of cells to a fresh 120 mL (final volume) of pre-warmed ExpiCHO TM The cell concentration was about 4 × 10 6 -6×10 6 Viable cells / mL;

[0138] (2) One day before transfection, place ExpiCHO-S TM The cells were diluted to a concentration of 3.5 × 10 6 viable cells / mL, cells were cultured overnight;

[0139] (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;

[0140] (4) Use fresh ExpiCHO preheated to 37°C TM Dilute the cells to 6 × 10 6 The calculated required volume of cells was transferred to a fresh 100 mL (final volume) of pre-warmed ExpiCHO TM in a 500 mL shake flask containing expression medium;

[0141] (5) Gently invert to mix ExpiFectamine TM CHO reagent, 3.7 mL OptiPRO TM Dilute ExpiFectamine in culture medium TM CHO reagent, swirl or mix;

[0142] (6) Use 4 mL of Refrigerated OptiPRO TM Dilute the plasmid DNA with culture medium and vortex to mix;

[0143] (7) Incubate the ExpiFectamine CHO / plasmid DNA (the plasmid DNA is the Fc fusion antibody eukaryotic expression vector of the anti-CD123 single domain antibody prepared in Example 6) complex at room temperature for 1-5 minutes, and then gently add it to the prepared cell suspension, gently shaking the flask during the addition process;

[0144] (8) The cells were cultured with shaking at 37°C, 8% CO2, and humidified air;

[0145] (9) On the first day after transfection (18-22 hours later), add 600 μl of ExpiFectamine TM CHO Enhancer and 24mLExpiCHO feed;

[0146] (10) Collect the supernatant about 8 days after transfection (cell viability is less than 70%).

[0147] Example 8: Expression of single domain antibodies against CD123 protein in suspension 293F cells

[0148] Recombinant single domain antibody expression experimental process (taking 500mL shake flask as an example):

[0149] (1) 3 days before transfection, 2.5×10 5 / mL cell passage and expansion culture of 293F cells, the calculated required cell volume was transferred to a 500mL shake flask filled with fresh pre-warmed 120mL (final volume) of OPM-293CD05 Medium. The cell concentration reached about 2×10 6 -3×10 6 Viable cells / mL.

[0150] (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.

[0151] (3) Dilute the cells to 1×10 using pre-warmed OPM-293CD05 Medium. 6 The calculated volume of cells was transferred to a 500 mL shake flask containing 100 mL (final volume) of fresh pre-warmed culture medium.

[0152] (4) Dilute PEI (1 mg / mL) reagent with 4 mL Opti-MEM medium, vortex or pipette to mix; dilute plasmid DNA (plasmid DNA is the Fc fusion antibody eukaryotic expression vector of the single domain antibody against CD123 prepared in Example 6) with 4 mL Opt-MEM medium, vortex to mix, and filter with a 0.22 um filter. Incubate at room temperature for 5 min.

[0153] (5) Add the diluted PEI reagent to the diluted DNA and mix by inverting. Incubate the PEI / plasmid DNA complex at room temperature for 15-20 minutes, then gently add it to the prepared cell suspension, gently swirling the flask during the addition process.

[0154] (6) The cells were cultured at 37°C, 5% CO2, and shaking at 120 rpm.

[0155] (7) Add 5 mL of OPM-CHO PFF05 feed at 24 h and 72 h after transfection.

[0156] (8) Collect the supernatant about 7 days after transfection (cell viability is less than 70%).

[0157] Example 9: Purification of single domain antibodies against CD123 protein

[0158] (1) The protein expression supernatant obtained in Example 7 or 8 was filtered through a 0.45 μm disposable filter to remove insoluble impurities;

[0159] (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;

[0160] (3) The filtrate is passed through the Protein A prepacked column at a flow rate of 1 mL / min. In this step, the target protein in the filtrate will bind to the filler;

[0161] (4) washing the impurity proteins bound to the column with low-salt and high-salt buffers;

[0162] (5) Elute the target protein bound to the column using a low pH buffer;

[0163] (6) The eluate was quickly added with a Tris-HCl solution at pH 9.0 to neutralize it;

[0164] (7) The neutralized protein solution is dialyzed and then subjected to SDS-PAGE analysis to determine that the protein purity is above 95% and the concentration is above 0.5 mg / mL, and then stored at low temperature for future use.

[0165] Example 10: Construction of single domain antibody eukaryotic expression vector

[0166] The target vector RJK-V4-hFC1, which is universal for nano-antibodies, is a modification of the company's commercial vector pCDNA3.4 (vector data link: https: / / assets.thermofisher.com / TFS-Assets / LSG / manuals / pcdna3_4_topo_ta_cloning_kit_man.pdf) based on Invitrogen, which was fused with the Fc segment in the heavy chain coding sequence of human IgG1. That is, the vector contains the hinge region (Hinge) CH2 and CH3 region of the IgG1 heavy chain. The specific modification plan is as follows:

[0167] (1) Select the restriction enzyme sites XbaI and AgeI on pcDNA3.4;

[0168] (2) introducing a multiple cloning site (MCS) and a 6×His tag at the 5′ and 3′ ends of the Fc fragment coding sequence respectively by overlapping PCR;

[0169] (3) amplifying the above fragment by PCR using a pair of primers with XbaI and AgeI restriction sites respectively;

[0170] (4) using restriction endonucleases XbaI and AgeI to digest pcDNA3.4 and the recombinant DNA fragments in (3), respectively;

[0171] (5) The digested vector and the inserted fragment are ligated with T4 ligase, and the ligated product is then transformed into Escherichia coli, amplified, and sequenced to obtain a recombinant plasmid.

[0172] Example 11: Expression and purification of tool antibody (Tab) targeting human CD123

[0173] In this article, Tab1: TPP-8987 (US2019365916A1); Tab4: talacotuzumab;

[0174] The sequence was entrusted to General Biosystems (Anhui) Co., Ltd. for mammalian cell expression system codon optimization and cloned into pcDNA3.1 vector. After resistance screening, plasmid-positive bacteria were selected for amplification, and plasmids were extracted using a plasmid extraction kit (Macherey Nagel, Cat#740412.50). 100 μg of plasmid (40 μg heavy chain + 60 μg light chain) was added per 100 mL of cells, and transient expression was performed in 293F cells (culture medium: FreeStyle 293 Expression medium, Thermo, Cat#12338026+F-68, Thermo, Cat#24040032) using PEI; 6 to 24 hours after transfection, 5% volume of 10% Peptone (Sigma, Cat#P0521-100G) was added, and cultured for about 7 to 8 days at 8% CO2 130rpm; when the cell viability dropped to 50%, the expression supernatant was collected and purified using ProteinA (GE, Cat#17-5438-02) gravity column; after dialysis with PBS, the concentration was determined using Nanodrop, the purity was identified by SEC, and the binding ability was verified by indirect ELISA;

[0175] The concentration of Tab1 and Tab4 obtained by the method is not less than 2 mg / ml and the purity is greater than 95%.

[0176] Example 12: Antibody antigen binding dose-effect curve determination

[0177] This example was performed using a standard enzyme-linked immunosorbent assay (ELISA) procedure.

[0178] (1) Coat with 50 μL of 1 μg / mL CD123 protein at 4°C overnight.

[0179] (2) Wash the plate; add 200 μL of 5% milk and block at 37°C for 2 h.

[0180] (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 protein of the single-domain antibody of the anti-CD123 protein obtained in Example 8 (expressed in 293F cells) by Example 9. In addition, hIgG, Tab1, and Tab4 controls are set respectively; Tab1 and Tab4 are prepared by Example 11; wherein hIgG refers to an isotype control, an immunoglobulin molecule that does not bind to any target, and is purchased commercially.

[0181] (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 h.

[0182] (5) Wash the plate; add 50 μL of HRP-Goat anti hIgG secondary antibody and incubate at 37°C for 30 min.

[0183] (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 min in the dark.

[0184] (7) Add 50 μL of stop solution (1N HCl) and read the result with a microplate reader.

[0185] (8) Draw the curve and calculate the EC50, such as Figure 1-Figure 5 Among them, 1A2, 1A5, 1A7, 1A8, 1A9, 1A10, 1A11, 1C2, 1C3, 1C8, 1D2, 1D8, 1E6, 1F12, 2B1, 2B6, 2B9, 2C6, and 2E6 are single domain antibodies whose sequences are not shown.

[0186] from Figure 1-Figure 5 It can be seen that the single-domain antibodies 1A6, 1F6, 2A9, 1A3, 1C10, 1D4, 1G7, and 1D1 all have strong specificity and affinity for CD123 protein.

[0187] Embodiment 13:

[0188] The ADCC effect of the single domain antibody of the present invention is determined by using a reporter gene method, and the steps are as follows:

[0189] (1) Tumor cells (CHO-K1-CD123) at passage 3-4 after recovery were collected and plated into 96-well plates at 20,000 cells per well;

[0190] (2) Tab, hIgG and VHH-hFc samples were prepared with a maximum concentration of 10 μg / mL solution and diluted 10 times to obtain 7 concentrations; VHH-hFc was obtained by purifying the Fc fusion protein of the single domain antibody of the anti-CD123 protein prepared in Example 8 (expressed in 293F cells) by Example 9.

[0191] (3) adding the gradient diluted antibody solution into the cell culture wells according to the equal volume of the cell suspension;

[0192] (4) For the sample wells and E / T wells (antibody concentration is 0), Jurkat-NFAT-luc-FcγRIIIa cells were collected and added to the cell culture wells at a rate of 20,000 cells per well;

[0193] (5) After 6 h of incubation, cell killing was detected using the One-Glo kit and luminescence was read;

[0194] (6) Calculate the fold of induction = (sample-BG) / (E / T-BG)

[0195] According to the target cell killing rate and concentration, four-parameter fitting was performed to calculate the EC50 concentration of ADCC mediated by each antibody, such as Figure 6-11 Among them, 1D8, 2B9, 2G11, 2B6, 1C2, 1C3, 1C8, 1D2, 1D8, 1E6, 1F6, and 1F12 are single domain antibodies whose sequences are not shown.

[0196] from Figure 6-Figure 11 It can be seen that the single-domain antibodies 1A6, 1F6, 2A9, 1A3, 1C10, 1D4, 1G7, and 1D1 all have ADCC effects.

[0197] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An antigen binding protein that binds to CD123, characterized in that: The antigen binding protein comprises a single domain antibody against CD123; the single domain antibody is composed of a heavy chain, and the heavy chain comprises a) a heavy chain CDR1 or a variant of CDR1 as shown in any one of SEQ ID NO:25 to SEQ ID NO:30, wherein the variant of CDR1 comprises a substitution of at most 2 amino acids in the CDR1; b) the heavy chain CDR2 or a variant of CDR2 as shown in SEQ ID NO: 31; the variant of CDR2 comprises a substitution of up to 2 amino acids in the CDR2; c) a heavy chain CDR3 or a variant of CDR3 as shown in any one of SEQ ID NO:32 to SEQ ID NO:33, wherein the variant of CDR3 comprises a substitution of at most 2 amino acids in the CDR3.

2. An antigen binding protein that binds to CD123 according to claim 1, characterized in that: The heavy chain includes a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3; the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 are one of the following (1)-(7): (1) CDR1 shown in SEQ ID NO:25, CDR2 shown in SEQ ID NO:31, and CDR3 shown in SEQ ID NO:32; (2) CDR1 shown in SEQ ID NO:25, CDR2 shown in SEQ ID NO:31, and CDR3 shown in SEQ ID NO:33; (3) CDR1 shown in SEQ ID NO:26, CDR2 shown in SEQ ID NO:31, and CDR3 shown in SEQ ID NO:32; (4) CDR1 shown in SEQ ID NO:27, CDR2 shown in SEQ ID NO:31, and CDR3 shown in SEQ ID NO:32; (5) CDR1 shown in SEQ ID NO:28, CDR2 shown in SEQ ID NO:31, and CDR3 shown in SEQ ID NO:32; (6) CDR1 shown in SEQ ID NO:29, CDR2 shown in SEQ ID NO:31, and CDR3 shown in SEQ ID NO:32; (7) CDR1 shown in SEQ ID NO:30, CDR2 shown in SEQ ID NO:31, and CDR3 shown in SEQ ID NO:

32.

3. An antigen binding protein that binds to CD123 according to claim 1, characterized in that: The heavy chain further comprises a framework region FR; the framework region FR comprises the amino acid sequences of FR1, FR2, FR3 and FR4, and the amino acid sequences of the framework region FR are respectively: FR1 or a variant of FR1 as shown in any one of SEQ ID NOs: 17-18, wherein the variant of FR1 comprises a substitution of up to 5 amino acids in FR1; FR2 or a variant of FR2 as shown in SEQ ID NO: 19, wherein the variant of FR2 comprises a substitution of up to 5 amino acids in FR2; FR3 or a variant of FR3 as shown in any one of SEQ ID NOs: 20-23, wherein the variant of FR3 comprises a substitution of up to 5 amino acids in FR3; FR4 or a variant of FR4 as shown in SEQ ID NO:24, wherein the variant of FR4 comprises a substitution of up to 5 amino acids in the FR4.

4. An antigen binding protein that binds to CD123, characterized in that: The antigen binding protein comprises a single domain antibody against CD123; the amino acid sequence of the single domain antibody is shown in any one of SEQ ID NOs: 1-8, or the amino acid sequence of the single domain antibody has at least 80% sequence homology with any one of SEQ ID NOs: 1-8.

5. An Fc fusion protein composed of an antigen binding protein that binds to CD123 according to any one of claims 1 to 4 and Fc, or a humanized antibody of an antigen binding protein that binds to CD123 according to any one of claims 1 to 4.

6. A recombinant protein, characterized in that The recombinant protein comprises the antigen-binding protein that binds to CD123 according to any one of claims 1 to 4.

7. A nucleotide molecule encoding an antigen binding protein that binds to CD123 according to any one of claims 1 to 4, characterized in that: The nucleotide sequence thereof is shown in any one of SEQ ID NOs: 9-16, or the nucleotide sequence has at least 80% sequence homology with any one of SEQ ID NOs: 9-16.

8. An expression vector, characterized in that: It comprises a nucleotide molecule encoding an antigen binding protein that binds to CD123 according to any one of claims 1 to 4, or an Fc fusion protein or a humanized antibody according to claim 5, or a nucleotide molecule according to claim 7.

9. A host cell, characterized in that: It can express the antigen binding protein binding to CD123 according to any one of claims 1 to 4 or the Fc fusion protein or humanized antibody according to claim 5, or it contains the expression vector according to claim 8.

10. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises an antigen binding protein that binds to CD123 selected from any one of claims 1 to 3, and a pharmaceutically acceptable carrier.

11. Use of the antigen binding protein that binds to CD123 according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 10 in the preparation of a medicament for treating a disease.

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