CN116715770A

CN116715770A - Single-domain antibody for resisting ephA2 and application thereof

Info

Publication number: CN116715770A
Application number: CN202310703114.1A
Authority: CN
Inventors: 苏志鹏; 王乐飞; 孟巾果; 张云
Original assignee: Nanjing Rongjiekang Biotechnology Co ltd
Current assignee: Nanjing Rongjiekang Biotechnology Co ltd
Priority date: 2023-06-14
Filing date: 2023-06-14
Publication date: 2023-09-08

Abstract

The application belongs to the field of immunology, and relates to an anti-ephA 2 single domain antibody and application thereof. The single domain antibody is composed of a heavy chain, wherein the heavy chain comprises a heavy chain CDR1 shown in any one of SEQ ID NO:43-SEQ ID NO:48, a heavy chain CDR2 shown in any one of SEQ ID NO:49-SEQ ID NO:54 and a heavy chain CDR3 shown in any one of SEQ ID NO:55-SEQ ID NO: 60. Compared with the prior art, the application has the beneficial effects that: the application uses biological gene engineering technology to screen out the single domain antibody specific to ephA2, and the antibody affinity is better.

Description

Single-domain antibody for resisting ephA2 and application thereof

Technical Field

The present application relates to a single domain antibody capable of specifically binding to ephA2 (hereinafter, abbreviated as "ephA2 single domain antibody"), a pharmaceutical composition containing the single domain antibody as an active ingredient, and a pharmaceutical therapeutic use thereof.

Background

The Eph receptors and the corresponding Eph receptor interaction (ephrin) ligands together constitute a critical cellular signaling network with multiple functions. Tyrosine kinase EphA2 belongs to the Eph receptor family, is abundantly produced in tumor tissues, and is found at relatively low levels in most normal adult tissues, suggesting its potential use in cancer therapy. Meanwhile, several EphA 2-targeting compounds have been evaluated and tested in clinical studies, despite limited clinical success.

EphA2, previously known as ECK, is a 130KD transmembrane receptor tyrosine kinase expressed in adult epithelial cells. See Lindberg et al (1990) mol.cell.biol.10:6316-6324.EphA2 is a member of the Eph family of receptor tyrosine kinases, which is unique in that they recognize ligands anchored to the cell membrane of adjacent cells, known as ephrins. See Bartley et al (1994) Nature368:558-560. The sequence of the human EphA2 receptor is known in the literature. It contains an extracellular domain of 534 amino acids, a transmembrane domain of 24 amino acids, and a cytoplasmic domain of 418 amino acids containing a tyrosine kinase domain.

EphA2 is overexpressed in a variety of cancer cells, e.g., in breast, prostate, lung, and colon cancers and invasive melanoma (aggressive melanomas), but there is no report of overexpression in non-cancerous lesions of these same tissues. See, for example, rosenberg et al (1997) am. J. Physiol.273:G824-G832; easty et al (1995) int.J.cancer 60:129-136; walker-Daniels et al (1999) Prostate 41:275-280; zantek et al (1999) Cell Growth & Differ.10:629-638; zantek et al (2001) Clin. Cancer rRs.7:3640-3648; zelniski et al (2001) Cancer Res.61:2301-2306; WO01/121172; and WO01/12840.

Disclosure of Invention

The object of the application of this patent is to provide a single domain antibody capable of specifically binding to ephA2 and its use.

In a first aspect, the application provides a single domain antibody against ephA2, said single domain antibody comprising a heavy chain CDR1 as shown in any one of SEQ ID NO:43-SEQ ID NO:48, a heavy chain as shown in any one of SEQ ID NO: 49-54

CDR2 and SEQ ID NO:55-SEQ ID NO: 60.

Preferably, the amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are one of the following (1) to (7):

(1) CDR1 as shown in SEQ ID NO. 43, CDR2 as shown in SEQ ID NO. 49, CDR3 as shown in SEQ ID NO. 58;

(2) CDR1 shown in SEQ ID NO. 44, CDR2 shown in SEQ ID NO. 52, CDR3 shown in SEQ ID NO. 56;

(3) CDR1 shown in SEQ ID NO. 44, CDR2 shown in SEQ ID NO. 51, CDR3 shown in SEQ ID NO. 56;

(4) CDR1 as shown in SEQ ID NO. 45, CDR2 as shown in SEQ ID NO. 54, CDR3 as shown in SEQ ID NO. 57;

(5) CDR1 shown in SEQ ID NO. 46, CDR2 shown in SEQ ID NO. 50, CDR3 shown in SEQ ID NO. 55;

(6) CDR1 shown in SEQ ID NO. 47, CDR2 shown in SEQ ID NO. 49, CDR3 shown in SEQ ID NO. 59;

(7) CDR1 shown in SEQ ID NO. 48, CDR2 shown in SEQ ID NO. 53, CDR3 shown in SEQ ID NO. 60.

The above CDR combinations (1) - (4) correspond to the single domain antibodies 1F7, 2A1, 2C6, 2C1 in sequence, CDR combination (5) corresponds to the single domain antibodies 1D8, 2A5, 2B7, 2E7, CDR combinations (6) - (7) correspond to the single domain antibodies 3A11, 3C1, respectively.

All of the above sequences may 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".

In one embodiment, wherein any one to five of the amino acid residues in any one or more of the CDRs of heavy chain CDR1, CDR2 and CDR3 may be substituted with their conserved amino acids, respectively. In particular, in the heavy chain CDR1, 1 to 5 amino acid residues may be replaced by their conserved amino acids; in the heavy chain CDR2, 1 to 5 amino acid residues may be replaced by their conserved amino acids; in the heavy chain CDR3, 1 to 5 amino acid residues may be replaced by their conserved amino acids.

As used herein, the term "sequence homology" refers to the degree 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 with identical sequences have 100% homology.

In some embodiments, sequences that replace only one or a few amino acids, e.g., comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, as compared to the preceding sequences, may also achieve the object. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminal and/or N-terminal end. In fact, the skilled person may consider so-called "conservative" amino acid substitutions, which in the case of substitution would preferably be conservative amino acid substitutions, in determining the degree of sequence homology between two amino acid sequences or in determining the CDR1, CDR2 and CDR3 combinations in a single domain antibody. The conserved amino acid, which may be generally described as an amino acid substitution of an amino acid residue with another amino acid residue having a similar chemical structure, has little or no effect on the function, activity, or other biological property of the polypeptide. Such conservative amino acid substitutions are common in the art, e.g., conservative amino acid substitutions are those in which one or a few amino acids in the following groups (a) - (d) are substituted for another or a few amino acids in the same group: (a) a polar negatively charged residue and an uncharged amide thereof: asp, asn, glu, gln; (b) a polar positively charged residue: his, arg, lys; (c) aromatic residues: phe, trp, tyr; (d) aliphatic nonpolar or low polar residues: ala, ser, thr, gly, pro, met, leu, ile, val, cys. Particularly preferred conservative amino acid substitutions are as follows: asp is substituted with Glu; asn is substituted with Gln or His; glu is substituted with Asp; gln is substituted with Asn; his is substituted with Asn or Gln; arg is replaced by Lys; lys is substituted by Arg, gln; phe is replaced by Met, leu, tyr; trp is substituted with Tyr; tyr is substituted with Phe, trp; substitution of Ala with Gly or Ser; ser is substituted by Thr; thr is replaced by Ser; substitution of Gly with Ala or Pro; met is substituted with Leu, tyr or Ile; leu is substituted with Ile or Val; lie is substituted with Leu or Val; val is substituted with Ile or Leu; cys is replaced by Ser. In addition, those skilled in the art will recognize that the creativity of single domain antibodies is represented in the CDR1-3 regions, while the framework region sequences FR1-4 are not immutable, and that the sequences of FR1-4 may take the form of conservative sequence variants of the sequences disclosed herein.

The meaning of "anti-ephA 2 single domain antibody" in the present application includes not only the whole single domain antibody but also fragments, derivatives and analogues of the anti-ephA 2 single domain antibody. As used herein, the terms "fragment," "derivative," and "analog" are synonymous and refer to a polypeptide that retains substantially the same biological function or activity of an antibody of the application. The polypeptide fragment, derivative or analogue of the application may be (i) a polypeptide having one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, substituted, which may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent 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, for example polyethylene glycol, or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence, such as a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with an Fc tag. Such fragments, derivatives and analogs are within the purview of one skilled in the art and would be well known in light of the teachings herein.

In a preferred embodiment, the antibody sequence further comprises a framework region FR; the framework regions FR include the amino acid sequences of FR1, FR2, FR3 and FR 4; the amino acid sequences of the framework regions FR are respectively:

21-27, said FR1 variant comprising up to 5 amino acid substitutions in said FR 1;

28-33, said FR2 variant comprising up to 5 amino acid substitutions in said FR 2;

34-39, said FR3 variant comprising up to 5 amino acid substitutions in said FR 3;

SEQ ID NO:40-42, or a variant of FR4 comprising up to 5 amino acid substitutions in said FR 4.

In a second aspect of the application there is provided an amino acid sequence of a single domain antibody capable of binding ephA2, said single domain antibody having the amino acid sequence set forth in SEQ ID NO:1-10, or said single domain antibody hybridizes to SEQ ID NO:1-10 and is capable of specifically binding to ephA2 protein, or the amino acid sequence of the single domain antibody has at least 80% sequence homology to SEQ ID NO:1-10, wherein at least 1 amino acid residue in the FR1, FR2, FR3 or FR4 sequence is substituted with a conserved amino acid.

In one embodiment, the anti-ephA 2 single domain antibody hybridizes to a polypeptide selected from the group consisting of SEQ ID NOs: 1-10 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology and is capable of specifically binding ephA2 protein.

In a third aspect of the application, there is provided an Fc fusion antibody or a humanized antibody of any one of the aforementioned single domain antibodies against ephA 2.

In a fourth aspect, the present application provides a nucleotide molecule encoding the aforementioned anti-ephA 2 single domain antibody or the aforementioned Fc fusion antibody or the aforementioned humanized antibody, having the nucleotide sequence set forth in SEQ ID NO:11-20, or the amino acid sequence encoded by said nucleotide sequence hybridizes with SEQ ID NO:11-20, or is identical to the amino acid sequence encoded by any one of SEQ ID NOs: 11-20 has at least 95% sequence homology.

In one embodiment, the nucleic acid molecule encoding the anti-ephA 2 single domain antibody hybridizes to a nucleic acid molecule selected from the group consisting of SEQ ID NO:11-20, and which encodes a single domain antibody against ephA2 capable of specifically binding to ephA2 protein, has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology.

In a fifth aspect, the present application provides an expression vector comprising a nucleotide molecule encoding a single domain antibody or Fc fusion antibody or humanized antibody against ephA2, having the nucleotide sequence set forth in SEQ ID NO:11-20 or the nucleotide sequence encodes an amino acid sequence that hybridizes with SEQ ID NO:11-20, and the amino acid sequence encoded by any one of the two.

In a preferred embodiment, the expression vector used is RJK-V4-3 (the nucleotide molecule encoding the anti-ephA 2 single domain antibody or its Fc fusion antibody or humanized antibody is integrated into RJK-V4-3 by genetic engineering means), and other general expression vectors may be selected as desired.

In a sixth aspect, the present application provides a host cell capable of expressing the aforementioned single domain antibody, fc fusion antibody or humanized antibody against ephA2, or comprising the aforementioned expression vector. Preferably the host cell is a bacterial cell, a fungal cell or a mammalian cell.

In another preferred embodiment, the host cell comprises a prokaryotic cell or a eukaryotic cell, including bacteria, fungi.

In another preferred embodiment, the host cell is selected from the group consisting of: coli, yeast cells, mammalian cells, phage, or combinations thereof.

In another preferred embodiment, the prokaryotic cell is selected from the group consisting of: coli, bacillus subtilis, lactobacillus, streptomyces, proteus mirabilis, or combinations thereof.

In another preferred embodiment, the eukaryotic cell is selected from the group consisting of: pichia pastoris, saccharomyces cerevisiae, schizosaccharomyces, trichoderma, or a combination thereof.

In another preferred embodiment, the eukaryotic cell is selected from the group consisting of: insect cells such as myxoplasma gondii, plant cells such as tobacco, BHK cells, CHO cells, COS cells, myeloma cells, or combinations thereof.

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

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

In a seventh aspect, the present application provides a recombinant protein comprising the aforementioned single domain antibody against ephA 2. The recombinant protein can be the aforementioned SEQ ID NO:1-10, or a single domain antibody as set forth in SEQ ID NO:1-10, and may also be a multi-epitope antibody, a multi-specific antibody, and a multivalent antibody; for example, the multi-epitope antibody can consist of SEQ ID NO:1-10, and a sequence of more than one of the sequences; the multivalent antibody can be represented by SEQ ID NO:1-10, wherein one sequence is repeatedly arranged for a plurality of times; such multispecific antibodies include, but are not limited to, the bispecific antibodies described above, as well as trispecific antibodies; furthermore, the recombinant proteins may be fragments, derivatives and analogues of the aforementioned antibodies.

An eighth aspect of the present application provides a pharmaceutical composition comprising the aforementioned ephA 2-binding single domain antibody and a pharmaceutically acceptable carrier. Typically, these materials are formulated in a nontoxic, 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 is required to deviate from and from about 2 from the isoelectric point of the antibody). The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intravenous and transdermal (directly applied or plastered on affected part).

The pharmaceutical compositions of the application contain a safe and effective amount (e.g., 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80 wt%) of the foregoing single domain antibodies, together with a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical compositions of the application may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical compositions, such as injections, solutions are preferably manufactured under sterile conditions.

In a ninth aspect, the present application provides an agent for treating a disease, which comprises the aforementioned single domain antibody for binding to ephA2 protein as an active ingredient.

In a tenth aspect, the present application provides a kit for detecting ephA2 levels, which comprises the aforementioned single domain antibody against ephA 2. In a preferred embodiment of the application, the kit further comprises a container, instructions for use, buffers, etc.

In a preferred embodiment, the kit comprises antibodies recognizing ephA2 protein, a lysis medium for lysing the sample, and universal reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, and the like. The detection kit may be an in vitro diagnostic device.

In a preferred embodiment, the kit further comprises a second antibody and an enzyme or fluorescent or radiolabel for detection, and a buffer.

In a preferred embodiment, the second antibody of the kit may be an antibody (as an anti-antibody) to the aforementioned single domain antibody of ephA2, and may be a single domain antibody, a monoclonal antibody, a polyclonal antibody, or any other form of antibody.

In an eleventh aspect of the present application, there is provided a method for producing a single domain antibody against ephA2, comprising the steps of:

(a) Culturing the host cell of the sixth aspect of the application under conditions suitable for the production of a single domain antibody, thereby

Obtaining a culture comprising said anti-ephA 2 single domain antibody; and

(b) Isolating or recovering said anti-ephA 2 single domain antibody from said culture; and

(c) Optionally, purifying and/or modifying the single domain antibody of ephA2 obtained in step (b).

In a twelfth aspect, the present application provides the use of the aforementioned anti-ephA 2 single domain antibody or the aforementioned pharmaceutical composition for the preparation of a medicament for the treatment of a disease.

In a preferred embodiment, the disease includes, but is not limited to, non-small cell lung cancer, gastrointestinal tumor, head and neck tumor, ovarian cancer, triple negative breast cancer, bladder urothelial cancer, pancreatic cancer, colorectal cancer, bladder cancer, esophageal cancer, lymphatic cancer, and glioblastoma.

Advantageous effects

Compared with the prior art, the application has the beneficial effects that:

(1) The single domain antibodies of the application are specific for ephA2 proteins having the correct spatial structure.

(2) The single domain antibody obtained by the application has flexible expression system selection, can be expressed in a prokaryotic system or a eukaryotic system of yeast cells or mammalian cells, has low expression cost in the prokaryotic expression system, and can reduce the post production cost.

(3) The single-domain antibody obtained by the application has simple reconstruction of the multi-combination form of the antibody, can obtain multivalent and multi-specific antibodies through simple serial connection in a genetic engineering mode, has low immune heterogeneity and can not generate stronger immune response under the condition of not undergoing humanized reconstruction.

(4) The application provides single domain antibodies with a broader range of affinities, ranging from nM to pM, which provide multiple options for later use of the antibodies without affinity maturation.

Drawings

In order to more clearly illustrate the technical solution of the present application, the drawings that are needed in the embodiments will be briefly described below, and it will be obvious to those skilled in the art that other drawings can be obtained from these drawings without inventive effort.

FIG. 1 is a library enrichment profile of the targeted ephA2 antibody screen of example 3;

FIG. 2 is a graph (1D 8) showing the measurement of the antibody antigen binding response curve in example 11;

FIG. 3 is a graph (1F 7) showing the measurement of the antibody-antigen binding response curve in example 11;

FIG. 4 is a graph (2A 1) showing the measurement of the antibody antigen binding response curve in example 11;

FIG. 5 is a graph (2A 5) showing the measurement of the antibody antigen binding response curve in example 11;

FIG. 6 is a graph showing the measurement of the antibody antigen binding response curve in example 11 (2C 1, 2B 7);

FIG. 7 is a graph (2C 6) showing the measurement of the antibody antigen binding response curve in example 11;

FIG. 8 is a graph (2E 7) showing the measurement of the antibody antigen binding response curve in example 11;

FIG. 9 is a graph (3A 11) showing the measurement of the antibody antigen binding response curve in example 11;

FIG. 10 is a graph (3C 1) showing the measurement of the antibody antigen binding response curve in example 11.

Detailed Description

The present application is described in further detail below with reference to examples to enable those skilled in the art to practice the same by referring to the description.

As used herein, a "single domain antibody" (sdAb, also called nanobody or VHH by the developer Ablynx) is well known to those skilled in the art. A single domain antibody is an antibody whose complementarity determining region is part of a single domain polypeptide. Thus, a single domain antibody comprises a single complementarity determining region (single CDR1, single CDR2, and single CDR 3). Examples of single domain antibodies are heavy chain-only antibodies (which naturally do not comprise light chains), single domain antibodies derived from conventional antibodies, and engineered antibodies.

The single domain antibodies may be derived from any species including mice, humans, camels, llamas, goats, rabbits, and cattle. For example, naturally occurring VHH molecules may be derived from antibodies provided by camelidae species (e.g. camels, dromedaries, llamas and dromedaries). Like whole antibodies, single domain antibodies are capable of selectively binding to a particular antigen. A single domain antibody may contain only the variable domains of an immunoglobulin chain, which domains have CDR1, CDR2 and CDR3, as well as framework regions.

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

The term "humanized antibody" refers to an antibody obtained by fusing the heavy chain variable region of a target antibody (e.g., an animal antibody) to the constant region of a human antibody, or by grafting the complementarity determining regions (CDR 1 to CDR3 sequences) of a target antibody into the variable region of a human antibody, or by subjecting a target antibody to amino acid mutation according to the characteristics of the framework regions (FR 1 to FR 4) of a human antibody. Humanized antibodies can be synthesized or site-directed mutagenesis.

In the present application, a single domain antibody against ephA2 can be obtained from a sequence having high sequence homology with the CDR1-3 disclosed in the present application. In some embodiments, sequences having "at least 80% homology" or "at least 85% homology", "at least 90% homology", "at least 95% homology", "at least 98% homology" to the sequences in SEQ ID No.1-10 may achieve the object of the application.

In some embodiments, the polypeptide that hybridizes to SEQ ID NO:1-10, e.g., comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, may also achieve the object of the application. In fact, in determining the degree of sequence homology between two amino acid sequences or in determining the CDR1, CDR2 and CDR3 combinations in a single domain antibody, the skilled person may consider so-called "conservative" amino acid substitutions, which in the case of substitution will preferably be conservative amino acid substitutions, which may 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 which substitution has little or no effect on the function, activity or other biological properties of the polypeptide. Such conservative amino acid substitutions are common in the art, e.g., conservative amino acid substitutions are those in which one or a few amino acids in the following groups (a) - (d) are substituted for another or a few amino acids in the same group: (a) a polar negatively charged residue and an uncharged amide thereof: asp, asn, glu, gln; (b) a polar positively charged residue: his, arg, lys; (c) aromatic residues: phe, trp, tyr; (d) aliphatic nonpolar or low polar residues: ala, ser, thr, gly, pro, met, leu, ile, val, cys. Particularly preferred conservative amino acid substitutions are as follows: asp is substituted with Glu; asn is substituted with Gln or His; glu is substituted with Asp; gln is substituted with Asn; his is substituted with Asn or Gln; arg is replaced by Lys; lys is substituted by Arg, gln; phe is replaced by Met, leu, tyr; trp is substituted with Tyr; tyr is substituted with Phe, trp; substitution of Ala with Gly or Ser; ser is substituted by Thr; thr is replaced by Ser; substitution of Gly with Ala or Pro; met is substituted with Leu, tyr or Ile; leu is substituted with Ile or Val; lie is substituted with Leu or Val; val is substituted with Ile or Leu; cys is replaced by Ser. In addition, those skilled in the art will recognize that the creativity of single domain antibodies is represented in the CDR1-3 regions, while the framework region sequences FR1-4 are not immutable, and that the sequences of FR1-4 may take the form of conservative sequence variants of the sequences disclosed herein.

Preferred host cells of the application are bacterial cells, fungal cells or mammalian cells.

The preparation method comprises the steps of preparing target protein and a truncated form of the target protein through a genetic engineering technology, immunizing an inner Mongolian alashan alpaca with the obtained antigen protein, obtaining peripheral blood lymphocytes or spleen cells of the alpaca after multiple immunization, recombining a camel source antibody variable region coding sequence into a phage display carrier through a genetic engineering mode, screening out a specific antibody aiming at the antigen protein through the phage display technology, and further detecting the binding capacity of the specific antibody and the antigen and application of the specific antibody in treatment of autoimmune diseases.

The above technical solutions will now be described in detail by way of specific embodiments:

example 1: preparation of human ephA2 recombinant extracellular domain protein:

the human recombinant extracellular domain protein used in the patent is obtained by self-expression and purification of a company, and the design scheme of an expression vector of the human recombinant ephA2 protein is as follows:

the coding sequence for EphA2, which is identified as nm_004431.4, was retrieved in NCBI and the resulting amino acid sequence was identified as np_004422.2.

The nucleotide sequence encoding the extracellular end of EphA2 (amino acids 1 to 530) was cloned into the vector pcdna3.4 by means of gene synthesis. And (3) carrying out Sanger sequencing on the constructed vector, comparing the original sequences, carrying out mass extraction on the recombinant plasmid after confirming no errors, removing endotoxin, and carrying out target protein expression and purification on transfected suspension 293F cells, wherein the purity reaches more than 90%, and meets the animal immunization requirement.

Example 2: construction of a single domain antibody library against ephA2 protein:

1mg of the recombinant ephA2 protein obtained by purification in example 1 was mixed with an equal volume of Freund's complete adjuvant, and an inner Mongolian Alexa alpaca was immunized once a week for a total of 7 continuous immunizations, and animals were immunized with 1mg of ephA2 protein in equal volumes with Freund's incomplete adjuvant for the total six other immunizations except for the first immunization, which was to intensively stimulate the camel to produce antibodies against ephA2 protein.

After the animal immunization is finished, 150mL of camel peripheral blood lymphocytes are extracted, and RNA of the cells is extracted. cDNA was synthesized using the extracted total RNA, and VHH (antibody heavy chain variable region) was amplified by a nested PCR reaction using the cDNA as a template.

Then the restriction enzymes are used for respectively enzyme cutting pMECS vector and VHH fragment, and then ligating the digested fragments to the vector. Electrotransformation of the ligated fragments into competent cells TG1, construction of phage display library of ephA2 protein and measurement of library capacity, library capacity size of about 1X 10 ⁹ At the same time, the correct insertion rate of the library at the fragment of interest was detected by colony PCR identification.

The results showed that after PCR amplification of 30 randomly selected colonies from the library, 29 clones amplified bands of predicted size and 1 clone amplified incorrectly, so the correct insertion was 29.times.30.times.100%. Apprxeq.96.7%.

Example 3: screening of Single Domain antibodies against ephA2 protein:

200. Mu.L of the recombinant TG1 cells of example 2 were cultured in 2 XTY medium, during which 40. Mu.L of helper phage VCSM13 was added to infect TG1 cells, and cultured overnight to amplify phage, the phage was precipitated the next day with PEG/NaCl, and the amplified phage was collected by centrifugation.

NaHCO diluted at 100mM pH8.3 ₃ 500. Mu.g of ephA2 protein coupled to an ELISA plate, and left overnight at 4℃while negative control wells (medium control) were established; the next day 200 μl of 3% skim milk was added and blocked at room temperature for 2h; after blocking was completed, 100. Mu.l of amplified phage library (approximately 2X 10 ¹¹ Individual phage particles), 1h at room temperature; after 1 hour of action, the unbound phage were washed off by washing 15 times with PBS+0.05% Tween-20.

Phage specifically binding to ephA2 protein was dissociated with trypsin at a final concentration of 25mg/mL, and E.coli TG1 cells in the logarithmic growth phase were infected, cultured at 37℃for 1 hour, phage were generated and collected for the next round of screening, and the same screening process was repeated for 1 round, and enrichment was gradually obtained.

When the enrichment multiple reaches more than 10 times, the enrichment effect is shown in figure 1.

In fig. 1, P/n=number of monoclonal bacteria grown after infection of TG1 bacteria by phage with positive Kong Xi removal from biopanning/number of monoclonal bacteria grown after infection of TG1 bacteria by phage with positive Kong Xi removal, which parameter increases gradually after enrichment occurs; I/E = total phage added to positive wells per round of biopanning/total phage removed from positive Kong Xi per round of biopanning, which parameter gradually approaches 1 after enrichment has occurred.

Example 4: screening of specific positive clones for ephA2 by phage enzyme-linked immunosorbent assay (ELISA):

screening was performed according to the screening method described in example 3 above for 2 rounds of screening against a single domain antibody against ephA2 protein, the phage enrichment factor against ephA2 protein was 10 or more, 384 single colonies were selected from positive clones obtained by screening after the end of screening, inoculated into 96-well plates containing 2×TY medium of 100. Mu.g/mL ampicillin, respectively, and a blank was set, and after culturing at 37℃to the logarithmic phase, IPTG was added at a final concentration of 1mM, and culturing was carried out at 28℃overnight.

Obtaining a crude extract antibody by using a permeation swelling method; release ephA2 recombinant protein to 100mM NaHCO pH8.3, respectively ₃ 100. Mu.g of protein was coated in an ELISA plate (ELISA plate) at 4℃overnight. Transferring 100 mu L of the obtained crude antibody extract to an ELISA plate added with antigen, and incubating for 1h at room temperature; washing unbound Antibody with PBST, adding 100 μl of Mouse Anti-HA tag Anti-body (HRP) (Mouse Anti-HA horseradish peroxidase labeled Antibody, thermo Fisher) diluted 1:2000, and incubating for 1h at room temperature; washing off unbound antibody with PBST, adding horseradish peroxidase chromogenic solution, reacting at 37deg.C for 15min, adding stop solution, and reading absorption value at 450nm wavelength on an enzyme-labeled instrument.

When the OD value of the sample hole is more than 5 times that of the control hole, judging that the sample hole is a positive cloning hole; the positive clone well was transferred to LB medium containing 100. Mu.g/mL ampicillin to extract plasmids and sequenced.

And analyzing the gene sequence of each clone strain according to sequence alignment software VectorNTI, and regarding strains with the same CDR1, CDR2 and CDR3 sequences as the same clone strain and strains with different sequences as different clone strains to finally obtain the single-domain antibody specific to the ephA2 protein.

The amino acid sequence of the antibody is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 structure, which forms the whole VHH. The obtained single-domain antibody recombinant plasmid can be expressed in a prokaryotic system, and finally the single-domain antibody protein is obtained.

The CDR and FR sequences of the 10 single domain antibodies are shown in tables 1-7, and the amino acid sequences and nucleotide sequences of the 10 single domain antibodies are shown in tables 8 and 9, respectively.

TABLE 1 CDR1 sequences of 10 antibodies

TABLE 2 CDR2 sequences of 10 antibodies

TABLE 3 CDR3 sequences of 10 antibodies

TABLE 4 FR1 sequences of 10 antibodies

TABLE 5 FR2 sequences of 10 antibodies

TABLE 6 FR3 sequences of 10 antibodies

TABLE 7 FR4 sequences of 10 antibodies

TABLE 8 amino acid sequences of 10 antibodies

TABLE 9 nucleic acid sequences of 10 antibodies

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Example 5: purification and expression of specific single domain antibodies to ephA2 protein in host bacteria E.coli

Plasmids of the different clones obtained by sequencing (pMECS-VHH) in example 4 were electrotransformed into E.coli HB2151 and plated onto LB+amp+glucose-containing culture plates, which were incubated overnight at 37 ℃; individual colonies were selected and inoculated in 5mL of LB medium containing ampicillin, and shake-cultured overnight at 37 ℃.

Inoculating 1mL of overnight culture strain into 330mLTB culture solution, shake culturing at 37deg.C until OD600nm reaches 0.6-0.9, adding 1M IPTG, shake culturing at 28deg.C overnight; centrifuging, collecting escherichia coli, and obtaining an antibody crude extract by using a permeation swelling method;

the single domain antibody was purified by nickel column affinity chromatography.

Example 6: construction of Fc fusion antibody eukaryotic expression vector of anti-ephA 2 single domain antibody

(1) Subcloning the target sequence obtained in example 4 into a eukaryotic expression vector: the antibodies selected in example 4 were subjected to Sanger sequencing to obtain their nucleotide sequences;

(2) Synthesizing the nucleotide sequence into a vector RJK-V4-3 designed and modified by the company by a sequence synthesis mode to obtain a recombinant eukaryotic expression vector, wherein the modification method of the vector is as described in example 10;

(3) Converting the recombinant eukaryotic expression vector constructed in the step (2) into DH5 alpha escherichia coli, culturing to extract plasmids, and removing endotoxin;

(4) Sequencing and identifying the extracted plasmid;

(5) The recombinant vector after confirmation was prepared for subsequent eukaryotic cell transfection and expression, and after expression of the Fc protein of VHH by the method of example 7 or 8, the above antibody was purified by the method of example 9.

Example 7: single domain antibodies against ephA2 protein expression in suspension ExpiCHO-S cells

(1) 3 days before transfection at 2.5X10 ⁵ ExpiCHO-S cell passage and expansion culture/mL ^TM The cells, calculated desired cell volume, were transferred to an ExpiCHO containing fresh pre-warmed 120mL (final volume) ^TM 500mL shake flask of expression medium; to achieve a cell concentration of about 4X 10 ⁶ -6×10 ⁶ Living cells/mL;

(2) One day prior to transfection, expiCHO-S was used ^TM Cell dilution concentration to 3.5X10 ⁶ Living cells/mL, allowing the cells to incubate overnight;

(3) The day of transfection, cell density and percent viable cells were determined. The cell density should reach about 7X 10 before transfection ⁶ -10×10 ⁶ Living cells/mL;

(4) Fresh ExpiCHO preheated to 37 ℃ ^TM Dilution of cells to 6X 10 in expression Medium ⁶ Each living cell/mL. The calculated desired cell volume was transferred to 100mL (final volume) of expcho filled with fresh pre-warmed ^TM 500mL shake flask of expression medium;

(5) Gently mixing the mixture with the mixture of the Expifectamine in a reverse manner ^TM CHO reagent with 3.7mL OptiPRO ^TM Dilution of Expifectamine in Medium ^TM CHO reagent, whipping or mixing;

(6) With refrigerated 4mL OptiPRO ^TM Diluting plasmid DNA with culture medium, and mixing;

(7) Incubating ExpiFectamine CHO/plasmid DNA (plasmid DNA is Fc fusion antibody eukaryotic expression vector of anti-ephA 2 single domain antibody prepared in example 6) complex for 1-5 min at room temperature, then adding gently to the prepared cell suspension, and gently agitating the flask during the addition;

(8) The cells were incubated at 37℃with 8% CO ₂ Shake culturing in humidified air;

(9) 600ul of Expiectamine was added on day 1 (18-22 hours post transfection) ^TM CHO enhancement and 24mL of expi CHO feed.

(10) Supernatants were collected about 8 days after transfection (cell viability below 70%).

Example 8: expression of single domain antibodies against ephA2 protein in suspension 293F cells

Recombinant single domain antibody expression experimental procedure (500 mL shake flask for example):

(1) 3 days before transfection at 2.5X10 ⁵ The cells were passaged/mL and expanded 293F cells, and the calculated desired cell volume was transferred to a 500mL shake flask containing fresh pre-warmed 120mL (final volume) OPM-293CD05 Medium. To achieve a cell concentration of about 2X 10 ⁶ -3×10 ⁶ Living cells/mL.

(2) The day of transfection, cell density and percent viable cells were determined. The cell density should reach about 2X 10 before transfection ⁶ -3×10 ⁶ Living cells/mL.

(3) Dilution of cells to 1X 10 with pre-warmed OPM-293CD05 Medium ⁶ Each living cell/mL. The calculated cell volume required was transferred to a 500mL shake flask containing fresh pre-warmed 100mL (final volume) of medium.

(4) Diluting PEI (1 mg/mL) reagent with 4mL of Opti-MEM culture medium, and stirring or blowing to mix uniformly; plasmid DNA (plasmid DNA is an Fc fusion antibody eukaryotic expression vector of the anti-ephA 2 single domain antibody prepared in example 6) was diluted with 4mL of Opt-MEM medium, mixed by vortexing, and filtered with a 0.22um filter head. Incubate at room temperature for 5min.

(5) Diluted PEI reagent was added to the diluted DNA and mixed upside down. PEI/plasmid DNA complexes were incubated for 15-20 minutes at room temperature and then gently added to the prepared cell suspension, during which time the shake flask was gently swirled.

(6) The cells were incubated at 37℃with 5% CO ₂ 、120rpmShake culture.

(7) 5mL OPM-CHO PFF05 feed was added 24h, 72h post transfection.

(8) Supernatants were collected about 7 days after transfection (cell viability below 70%).

Example 9: purification of Single-Domain antibodies against ephA2 protein

(1) The protein expression supernatant obtained in example 7 or 8 was filtered with a disposable filter head of 0.45 μm to remove insoluble impurities;

(2) Purifying the filtrate by using a Protein purifier to perform affinity chromatography, and purifying by using agarose filler coupled with Protein A by utilizing the binding capacity of human Fc and Protein A;

(3) Passing the filtrate through a Protein A pre-packed column at a flow rate of 1 mL/min, wherein the target Protein in the filtrate is combined with the packing;

(4) Washing the column-bound impurity proteins with a low-salt and high-salt buffer;

(5) The target protein combined on the column is subjected to a system by using a low pH buffer solution;

(6) Rapidly adding the eluent into Tris-HCl solution with pH of 9.0 for neutralization;

(7) And (3) dialyzing the neutralized protein solution, performing SDS-PAGE analysis to determine that the protein purity is above 95%, and preserving the protein at a low temperature for later use after the concentration is above 0.5 mg/mL.

Example 10: construction of Single-Domain antibody eukaryotic expression vector RJK-V4-3

The mentioned nanobody universal targeting vector RJK-V4-3 is modified by the company after fusion of Fc region in heavy chain coding sequence of human IgG4 based on the invitrogen commercial vector pCDNA3.4 (vector data link: https:// assems. Thermo-former. Com/TFS-assems/LSG/manals/pcdna3_4_topo_ta_cloning_kit_man. Pdf), i.e. the vector comprises Hinge region (Hinge) CH2 and CH3 region of IgG4 heavy chain. The concrete improvement scheme is as follows:

(1) Selecting restriction enzyme cutting sites XbaI and AgeI on pcDNA3.4;

(2) Introducing multiple cloning sites (MCS, multiple Cloning Site) and a 6 XHis tag at the 5 'end and the 3' end of the coding sequence of the Fc fragment respectively by means of overlapping PCR;

(3) Amplifying the fragments by PCR using a pair of primers with XbaI and AgeI cleavage sites, respectively;

(4) The recombinant DNA fragments in pcDNA3.4 and (3) were digested with restriction enzymes XbaI and AgeI, respectively;

(5) And (3) connecting the digested vector and the inserted fragment under the action of T4 ligase, then converting the connection product into escherichia coli, amplifying, and checking by sequencing to obtain the recombinant plasmid.

Example 11: antigen binding quantitative profile assay for antibodies

This example was performed using standard enzyme-linked immunosorbent assay (ELISA) protocols.

(1) 50. Mu.L of 1. Mu.g/mL human ephA2 protein was coated overnight at 4 ℃.

(2) Washing the plate; 200. Mu.L of 5% milk was added and blocked at 37℃for 2h.

(3) VHH was diluted to 2ug/mL and then the antibody was diluted 5-fold gradient for a total of 8 concentration gradients. VHH herein refers to the prokaryotic-expressed single domain antibody against ephA2 protein prepared in example 5.

(4) Washing the plate; add 50. Mu.L of single domain antibody diluted in step (3), double wells and incubate at 37℃for 1h.

(5) Washing the plate; 50. Mu.L of HRP-coat anti-hIgG secondary antibody was added and incubated at 37℃for 30min.

(6) Washing the plate (washing several times); 50. Mu.L of TMB which had previously recovered the room temperature was added thereto, and the reaction was continued at the normal temperature in the dark for 15 minutes.

(7) Add 50. Mu.L of stop solution (1N HCl) and store the microplate reader reading.

(8) The EC50 was calculated by plotting a curve as shown in fig. 2-10, wherein igg designates a type control, immunoglobulin molecules that do not bind to any target, and are commercially available. Wherein the sequence of the single domain antibody 1D9, 1E1, 1E12, 1E2, 1E3, 1E9, 1F8, 1G2, 1G6, 1H10, 1H11, 1H7, 1H9, 2a10, 2a11, 2A7, 2B1, 2B10, 2B2, 2B4, 2B8, 2B9, 2C11, 2C12, 2C2, 2C4, 2C5, 2C9, 2D1, 2D2, 2D4, 2E10, 2E6, 2F11, 2F4, 2G4, 2H11, 2H6, 3B12, 3B2, 3B7, 3C3, 3C5, 3C1-2 is not shown in the present specification.

As can be seen from FIGS. 2 to 9, the single domain antibodies 1F7, 2A1, 2C6, 2C1, 1D8, 2A5, 2B7, 2E7, 3A11, 3C1 of the present application have a good affinity and a strong specificity for ephA2 protein.

The foregoing has shown and described the basic principles, principal features and advantages of the application. It will be appreciated by persons skilled in the art that the above embodiments are not intended to limit the application in any way, and that all technical solutions obtained by means of equivalent substitutions or equivalent transformations fall within the scope of the application.

Claims

1. A single domain antibody against ephA2, characterized in that: the single domain antibody is composed of a heavy chain, wherein the heavy chain comprises SEQ ID NO:

heavy chain CDR1 shown in any one of 43-SEQ ID NO:48, heavy chain shown in any one of SEQ ID NO:49-SEQ ID NO:54

CDR2 and SEQ ID NO:55-SEQ ID NO: 60.

2. The single domain antibody of ephA2 according to claim 1, wherein: 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):

3. The single domain antibody against ephA2 according to claim 1, characterized in that: the single domain antibody also comprises a framework region FR; the framework regions FR include the amino acid sequences of FR1, FR2, FR3 and FR 4; the amino acid sequences of the framework regions FR are respectively:

40-42, said FR4 variant comprising up to 5 amino acid substitutions in said FR 4.

4. A single domain antibody against ephA2, characterized in that: the amino acid sequences of the single-domain antibodies are respectively shown in SEQ ID NO:1-10, or the amino acid sequence of the single domain antibody hybridizes to SEQ ID NO:1-10, wherein at least 1 amino acid residue in the FR1, FR2, FR3 or FR4 sequence is substituted with a conserved amino acid.

5. The Fc fusion antibody or humanized antibody of the single domain antibody against ephA2 of any one of claims 1-4.

6. A recombinant protein comprising the single domain antibody of any one of claims 1 to 4 against ephA 2.

7. A nucleotide molecule encoding the single domain antibody against ephA2 according to any one of claims 1 to 4, characterized in that: the nucleotide sequences of the nucleotide sequences are respectively shown in SEQ ID NO:11-20, or the amino acid sequence encoded by said nucleotide sequence hybridizes with the amino acid sequence set forth in any one of SEQ ID NOs: 11-20, and the amino acid sequence encoded by any one of the two.

8. An expression vector, characterized in that: comprising a nucleotide molecule encoding the single domain antibody against ephA2 of any one of claims 1 to 4 or the Fc fusion antibody or humanized antibody of claim 5 or the nucleotide molecule of claim 7.

9. A host cell, characterized in that: which can express the single domain antibody against ephA2 of any one of claims 1 to 4 or the Fc fusion antibody or humanized antibody of claim 5, or which comprises the expression vector of claim 8.

10. A pharmaceutical composition characterized by: the pharmaceutical composition comprising a single domain antibody against ephA2 selected from any one of claims 1 to 4, and a pharmaceutically acceptable carrier.

11. A medicament for treating a disease characterized by: comprising the single domain antibody against ephA2 according to any one of claims 1 to 4 as an active ingredient.

12. Use of the single domain antibody of anti-ephA 2 according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 10 in the manufacture of a medicament for the treatment of a disease.

13. Use according to claim 12, characterized in that: such diseases include non-small cell lung cancer, gastrointestinal tumors, head and neck tumors, ovarian cancer, triple negative breast cancer, bladder urothelial cancer, pancreatic cancer, colorectal cancer, bladder cancer, esophageal cancer, lymphatic cancer, and glioblastoma.