CN116621984A

CN116621984A - anti-CD 155 single domain antibody and application thereof

Info

Publication number: CN116621984A
Application number: CN202310441274.3A
Authority: CN
Inventors: 苏志鹏; 孟巾果; 王乐飞; 张云
Original assignee: Nanjing Rongjiekang Biotechnology Co ltd
Current assignee: Nanjing Rongjiekang Biotechnology Co ltd
Priority date: 2023-04-23
Filing date: 2023-04-23
Publication date: 2023-08-22

Abstract

The invention belongs to the field of immunology, and relates to a single-domain antibody for resisting CD155 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. 25-SEQ ID NO. 29, a heavy chain CDR2 shown in any one of SEQ ID NO. 30-SEQ ID NO. 32 and a heavy chain CDR3 shown in any one of SEQ ID NO. 33-SEQ ID NO. 37. Compared with the prior art, the invention has the beneficial effects that: the invention uses biological gene engineering technology to screen out the single domain antibody specific to CD155, which has better affinity.

Description

anti-CD 155 single domain antibody and application thereof

Technical Field

The present invention relates to a single domain antibody capable of specifically binding to CD155 (hereinafter, abbreviated as "CD155 single domain antibody"), a pharmaceutical composition containing the single domain antibody as an active ingredient, and a pharmaceutical therapeutic use thereof.

Background

Cluster of differentiation 155 (CD 155) is a single transmembrane cell surface protein that is expressed little or not in normal tissues, but is highly expressed in a variety of malignant tumors including skin adenocarcinoma, cholangiocarcinoma, colorectal carcinoma, non-small cell lung carcinoma, bladder carcinoma, breast carcinoma, making it an ideal target for antitumor drugs, CD155 as a cell adhesion molecule involved in adhesion, migration and polarization of tumor cells, and as an immunomodulatory molecule interacting with co-stimulatory or co-inhibitory receptors on T cells or NK cells including TIGIT, DNA-1, CD96, exerting an immunomodulatory effect affecting the immune microenvironment.

The earliest drug development for targeting CD155 to treat tumors is to directly target the tumor cells which overexpress CD155 through the recombinant oncolytic poliovirus which effectively infects the tumor cells, so that the tumor cells are dissolved and cells die, and the immunoregulation effect is further exerted. With the progressive penetration of CD155 function, the targeted blocking of CD155 function as an immune checkpoint inhibitor, through the blocking of inhibitory interactions of immune cells in the tumor microenvironment by antibodies, reverses immunosuppression, increases Tumor Infiltrating Lymphocyte (TIL) activation and cytotoxicity, and ultimately leads to tumor cell death. Or blocking tumor cells with high expression of CD155 with monoclonal antibody, and inhibiting tumor cell development and metastasis. In addition, receptors that can target CD155 block immune checkpoint inhibition pathways.

Nanobodies are a new proposition in the antibody community, and because of the small molecular weight, bivalent, trivalent or bispecific antibodies can be obtained by simple molecular cloning techniques. Because of the characteristic of small molecules, the nanobody can achieve high yield in both prokaryotic expression systems (escherichia coli) and eukaryotic expression systems (CHO cells, 293 cells and the like). The rapid development of nanobodies has become a potential-unlimited force in antibody drug development, representing an important development direction of antibody drugs from now on.

Disclosure of Invention

The invention of this patent aims to provide a single domain antibody capable of specifically binding to CD155 and uses thereof.

In a first aspect the invention provides a single domain antibody against CD155, said single domain antibody comprising a heavy chain CDR1 as shown in any one of SEQ ID NO 25-SEQ ID NO 29, a heavy chain as shown in any one of SEQ ID NO 30-SEQ ID NO 32

CDR2 and SEQ ID NO 33-SEQ ID NO 37.

Preferably, the amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are one of the following (1) to (5):

(1) CDR1 shown in SEQ ID NO. 25, CDR2 shown in SEQ ID NO. 32, CDR3 shown in SEQ ID NO. 37;

(2) CDR1 shown in SEQ ID NO. 26, CDR2 shown in SEQ ID NO. 30, CDR3 shown in SEQ ID NO. 34;

(3) CDR1 shown in SEQ ID NO. 27, CDR2 shown in SEQ ID NO. 31, CDR3 shown in SEQ ID NO. 35;

(4) CDR1 shown in SEQ ID NO. 28, CDR2 shown in SEQ ID NO. 31, CDR3 shown in SEQ ID NO. 33;

(5) CDR1 shown in SEQ ID NO. 29, CDR2 shown in SEQ ID NO. 31, and CDR3 shown in SEQ ID NO. 36.

The above 5 CDR combinations (1) - (5) correspond in sequence to single domain antibodies 2F1, 1D4, 3B12, 2G4, 2C 1.

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-CD 155 single domain antibody" in the present invention includes not only the whole single domain antibody but also fragments, derivatives and analogues of the anti-CD 155 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 invention. The polypeptide fragment, derivative or analogue of the invention 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:

11-14, or a variant of FR1 as set forth in any one of SEQ ID nos. 11-14, said variant of FR1 comprising up to 5 amino acid substitutions in said FR 1;

15-17, said FR2 variant comprising up to 5 amino acid substitutions in said FR 2;

18-22, said FR3 variant comprising up to 5 amino acid substitutions in said FR 3;

23 or 24, said FR4 variant comprising up to 5 amino acid substitutions in said FR 4.

In a second aspect of the invention there is provided an amino acid sequence of a single domain antibody capable of binding CD155, said single domain antibody having the amino acid sequence shown in SEQ ID NO.1-5, respectively, or said single domain antibody having at least 80% sequence homology with the amino acid sequence of SEQ ID NO.1-5 and being capable of specifically binding CD155 protein.

In a preferred embodiment, the amino acid sequence of the single domain antibody hybridizes to SEQ ID NO:1-5, 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-CD 155 single domain antibody hybridizes to a polypeptide selected from the group consisting of SEQ ID NOs: 1-5 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology and is capable of specifically binding CD155 protein.

A third aspect of the invention is to provide an Fc fusion antibody or a humanized antibody of any one of the foregoing anti-CD 155 single domain antibodies.

In a fourth aspect, the present invention provides a nucleotide molecule encoding the aforementioned anti-CD 155 single domain antibody or the aforementioned Fc fusion antibody or the aforementioned humanized antibody, having the nucleotide sequence set forth in SEQ ID NO:6-10, or the amino acid sequence encoded by said nucleotide sequence hybridizes with SEQ ID NO:6-10, or is identical to the amino acid sequence encoded by any one of SEQ ID NOs: 6-10 has at least 95% sequence homology.

In one embodiment, the nucleic acid molecule encoding said anti-CD 155 single domain antibody hybridizes to a nucleic acid molecule selected from the group consisting of SEQ ID NOs: 6-10 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology and encodes a single domain antibody against CD155 capable of specifically binding to CD155 protein.

In a fifth aspect, the present invention provides an expression vector comprising a nucleotide molecule encoding a single domain antibody or an Fc fusion antibody or a humanized antibody against CD155, having the nucleotide sequence set forth in SEQ ID NO: 6-10.

In a preferred embodiment, the expression vector used is RJK-V4-hFC (the nucleotide molecules encoding the anti-CD 155 single domain antibody or its Fc fusion antibody or humanized antibody are integrated into RJK-V4-hFC by genetic engineering means), and other universal expression vectors may be selected as desired.

In a sixth aspect, the invention provides a host cell capable of expressing the foregoing single domain antibody, fc fusion antibody or humanized antibody against CD155, or comprising the foregoing 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 invention provides a recombinant protein comprising the foregoing anti-CD 155 single domain antibody. The recombinant protein can be a single-domain antibody shown in SEQ ID No.1-5, a single-domain antibody with at least 80% homology with SEQ ID No.1-5, a multi-epitope antibody, a multi-specific antibody and a multivalent antibody; for example, the multi-epitope antibody may consist of more than one of the sequences set forth in SEQ ID NOS.1-5; the multivalent antibody can be formed by repeatedly arranging one sequence in SEQ ID NO.1-5 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 invention provides a pharmaceutical composition comprising a single domain antibody that binds CD155 as described above 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 invention 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 invention 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 invention provides a pharmaceutical agent for treating a disease, comprising the aforementioned single domain antibody for binding to CD155 protein as an active ingredient.

In a tenth aspect, the invention provides a kit for detecting the level of CD155, which comprises the single domain antibody of CD 155. In a preferred embodiment of the invention, the kit further comprises a container, instructions for use, buffers, etc.

In a preferred embodiment, the kit comprises antibodies recognizing CD155 protein, a lysis medium for lysing the sample, universal reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, etc. 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 against CD155, may be a single domain antibody, a monoclonal antibody, a polyclonal antibody or any other form of antibody.

In an eleventh aspect of the invention, there is provided a method of producing a single domain antibody against CD155 comprising the steps of:

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

Obtaining a culture comprising said anti-CD 155 single domain antibody; and

(b) Isolating or recovering said anti-CD 155 single domain antibody from said culture; and

(c) Optionally purifying and/or modifying the single domain antibody of CD155 obtained in step (b).

In a twelfth aspect, the invention provides the use of the aforementioned anti-CD 155 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 comprises solid tumors, bladder cancer, brain tumors, head and neck cancer, breast cancer, rectal cancer, prostate cancer, glioblastoma, melanoma, head and neck squamous cell carcinoma, diabetic nephropathy, diabetic retinopathy and nonalcoholic steatohepatitis.

Advantageous effects

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

(1) The single domain antibodies of the invention are specific for CD155 protein with the correct spatial structure.

(2) The single domain antibody obtained by the invention 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 single domain antibody obtained by the application has wider affinity range, and the affinity range can be from nM level to pM level before affinity maturation, so that multiple choices are provided for antibodies with different later uses.

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 CD155 antibody screen of example 3;

FIG. 2 is a graph (1D 4) showing the measurement of the antibody-antigen binding response curve in example 12;

FIG. 3 is a graph (2C 1) showing the measurement of the antibody-antigen binding response curve in example 12;

FIG. 4 is a graph (2F1,2G4) showing the measurement of the antibody-antigen binding response curve in example 12;

FIG. 5 is a graph (3B 12) showing the measurement of the antibody antigen binding response curve in example 12;

FIG. 6 is a graph showing the measurement of the antibody antigen binding response curve in example 12 (Tab 1, tab2, hIgG);

FIG. 7 shows the results of ADCC effect detection of specific single domain antibodies to CD155 protein (Tab 1, tab2, hIgG, 3B 12);

FIG. 8 shows the results of ADCC effect detection of a specific single domain antibody of CD155 protein (1D 4, 2C1, 2F1, 2G 4).

Detailed Description

The present invention 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 CDR 3 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 invention, a single domain antibody against CD155 can be obtained even from a sequence having high sequence homology with the CDRs 1-3 disclosed in the present invention. 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.1-5 may achieve the object of the invention.

In some embodiments, the polypeptide that hybridizes to SEQ ID NO:1-5, 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 invention. 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 invention 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 CD155 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 CD155 protein is as follows:

(1) The coding sequence for CD155, designated NM-006505.4, was retrieved from NCBI and encoded to give the amino acid sequence accession NP-006496.4.

(2) The nucleotide sequence encoding the 1 st to 343 rd amino acids of the extracellular end of CD155 is 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 CD155 protein:

1mg of the recombinant human CD155 protein obtained by purification in example 1 was mixed with an equal volume of Freund's complete adjuvant, and an inner Mongolian Alexal camel was immunized once a week for 7 consecutive immunizations, and the remaining six immunizations were animal immunized with 1mg of CD155 protein mixed with Freund's incomplete adjuvant equal volume except for the first immunization, which was to intensively stimulate the camel to produce antibodies against CD155 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 pMECS vector and the VHH fragment were digested separately using restriction enzymes, and the digested fragments and vector were ligated. Electrotransformation of the ligated fragments into competent cells TG1, construction of a phage display library of CD155 protein and measurement of the library capacity, which was approximately 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.97%.

Example 3: single domain antibody screening against CD155 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 μg of CD155 protein coupled to an ELISA plate, and left overnight at 4deg.C 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.

The phage specifically binding to CD155 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 CD155 by phage enzyme-linked immunosorbent assay (ELISA):

screening was performed for 2 rounds of screening against single domain antibodies against CD155 protein according to the screening method described in example 3 above, phage enrichment factor against CD155 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 XSTY medium of 100. Mu.g/mL ampicillin, respectively, and a blank control was set, and after culturing at 37℃to 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; the CD155 recombinant protein was released 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 sequences of all clone strains according to sequence comparison software VectorNTI, and regarding the strains with the same CDR1, CDR2 and CDR3 sequences as the same clone strain and the strains with different sequences as different clone strains to finally obtain the single-domain antibody specific to the CD155 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 5 single domain antibodies are shown in tables 1-7, and the amino acid sequences and nucleotide sequences of the 5 single domain antibodies are shown in tables 8 and 9, respectively.

TABLE 15 CDR1 sequences of antibodies

TABLE 2 5 CDR2 sequences of antibodies

TABLE 3 5 CDR3 sequences of antibodies

Actual clone numbering	CDR3	SEQ ID
			2G4	AAARNGLWRRRESYKD	SEQ ID NO:33
1D4	AAARNGLWRRRESYNS	SEQ ID NO:34
			3B12	AAARNGLWRRRESYNY	SEQ ID NO:35
2C1	AAARNGSWRRRESYNY	SEQ ID NO:36
			2F1	AAGRSGTWRWANAYNY	SEQ ID NO:37

TABLE 4 5 FR1 sequences of antibodies

TABLE 55 FR2 sequences of antibodies

TABLE 6 5 FR3 sequences of antibodies

TABLE 7 5 FR4 sequences of antibodies

Table 8 5 amino acid sequences of antibodies

Table 9 5 nucleic acid sequences of antibodies

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Example 5: purification and expression of specific single domain antibody of CD155 protein in host bacterium escherichia 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 antibodies were purified by nickel column affinity chromatography and the purified fractions of single domain antibodies, including 1D4, 2C1, 2F1, 2G4, 3B12.

Example 6: construction of Fc fusion antibody eukaryotic expression vector of anti-CD 155 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-hFC designed and modified by the company in 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 CD155 protein are expressed 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-CD 155 single domain antibody prepared in example 6) complex for 1-5 minutes at room temperature, then adding gently into the prepared cell suspension, and gently agitating shake 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 anti-CD 155 protein Single Domain antibodies 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 293F cells were passaged and expanded per mL of cells, and the calculated desired cell volume was transferred to 500mL of shaking with fresh pre-warmed 120mL (final volume) OPM-293CD05 MediumIn a bottle. 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-CD 155 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 Shake culturing at 120 rpm.

(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 anti-CD 155 protein single domain antibodies

(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-hFC

The mentioned nanobody universal targeting vector RJK-V4-hFC was modified by the company after fusion of the Fc region in the heavy chain coding sequence of human IgG1 on the basis of the invitrogen commercial vector pCDNA3.4 (vector data link: https:// packages. Thermofiser. Com/TFS-packages/LSG/manual/pcdna3_4_topo_ta_cloning_kit_man. Pdf), i.e.the vector comprises the Hinge region (Hinge) CH2 and CH3 regions of the IgG1 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: expression and purification of Tool antibodies (Tabs) targeting human CD155

Here, tab1 is 4e5 in CN109071666a, and Tab2 is 7D4 in CN109071666 a. The searched sequences were commissioned for mammalian cell expression system codon optimization by general biosystems (Anhui) Inc., and cloned into pcDNA3.1 vector. Selecting a substance through resistance screeningPlasmid positive bacteria were amplified and plasmids were extracted using a plasmid extraction kit (Macherey Nagel, cat# 740412.50). According to the addition of 100. Mu.g of plasmid per 100mL of cells (40. Mu.g of heavy chain+60. Mu.g of light chain), PEI was transiently expressed in 293F cells (medium: freeStyle293Expression medium, thermo, cat #12338026+F-68, thermo, cat # 24040032); after 6-24 h of transfection 5% by volume of 10% Peptone (Sigma, cat#P0521-100G), 8% CO were added ₂ Culturing at 130rpm for about 7-8 days; when the cell viability was reduced to 50%, the expression supernatant was collected and purified using a gravity column of ProteinA (GE, cat#17-5438-02); after PBS dialysis, concentration was determined using Nanodrop, SEC to identify purity, and indirect ELISA to verify binding capacity;

tab obtained by the method has the concentration of not less than 2mg/ml and the purity of more than 95 percent.

Example 12: 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 CD155 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-hFc was diluted to 2ug/mL and then the antibody was diluted 5-fold gradient for a total of 8 concentration gradients. The VHH-hFc here refers to the Fc fusion antibody (expressed in 293F cells) of the single domain antibody against CD155 protein prepared in example 8, which was purified in example 9. In addition, hIgG and Tab controls are also respectively arranged; tab1, tab2 was prepared from example 11;

(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. 2-6, wherein igg designates a type control, immunoglobulin molecules that do not bind to any target, and are commercially available.

FIGS. 2 to 6 show the results of measurement of antigen binding capacity curves of samples (including 1D4, 2C1, 2F1, 2G4 and 3B12, and antibody strains not shown in the sequences: 1C10, 1C11, 1D10, 2A2, 2A3, 2A4, 2A11, 2B4, 2B8, 2C3, 2C8, 2E1, 2F8, 2F9, 2G7, 3A11, 3C4, 3C12 and 3F 3).

The single domain antibodies 1D4, 2C1, 2F1, 2G4 and 3B12 all exhibited excellent binding to CD155 antigen with better affinity and specificity than the hIgG and Tab controls.

Example 13: ADCC effect detection of specific single domain antibodies to CD155 protein

The purpose of this example was to perform ADCC effect detection on Fc fusion single domain antibodies specific for CD155, which were obtained by purification of Fc fusion antibodies (expressed in 293F cells) of single domain antibodies against CD155 protein prepared in example 8, using MDA-MB-231 cells, ADCC referring to antibody-dependent cell-mediated cytotoxicity. The experimental procedure was as follows:

(1) Collecting the MDA-MB-231 cells of 3-4 generations after resuscitating, and paving the collected MDA-MB-231 cells into a 96-well plate according to 10000 holes;

(2) Preparing solution with the highest concentration of 10 mug/mL by using Tab1, tab2, hIgG and antibody sample VHH-hFc, and carrying out 10-time gradient dilution to finally obtain 7 concentrations;

(3) Adding the antibody solution diluted in a gradient manner into a cell culture hole according to the equal volume of the cell suspension;

(4) For sample wells and E/T wells (antibody concentration 0), PBMC cells were collected and added to the cell culture wells at 250000 cells per well, twice the volume of the target cell suspension; for MAX wells, two times the volume of target cell suspension per well was added; for MIN wells, twice the volume of the target cell suspension was added to each well;

(5) After 6h incubation, detecting cell killing by using an LDH kit, and reading absorbance;

(6) Target cell killing% = (sample-E/T)/(MAX-MIN) according to the formula;

(7) Based on target cell killing rate and concentration, four parameter fits were performed to calculate EC50 concentrations for each antibody-mediated ADCC.

After analysis by established standard curves, the detection results are shown in FIGS. 7 to 8 (including 1D4, 2C1, 2F1, 2G4 and 3B12, and antibody strains 3H7, 2A4, 2B8, whose sequences are not shown). It can be seen that the single domain antibodies 1D4, 2C1, 2F1, 2G4 and 3B12 all have better ADCC effect than the hIgG and Tab controls.

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

Claims

1. A single domain antibody against CD155, characterized in that: the single domain antibody is composed of a heavy chain, wherein the heavy chain comprises SEQ ID NO:

heavy chain CDR1 as shown in any one of 25-SEQ ID NO. 29, heavy chain CDR2 as shown in any one of SEQ ID NO. 30-SEQ ID NO. 32 and heavy chain CDR3 as shown in any one of SEQ ID NO. 33-SEQ ID NO. 37.

2. The single domain antibody of CD155 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) - (5):

3. The anti-CD 155 single domain antibody of claim 1, wherein: 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:

4. A single domain antibody against CD155, characterized in that: the amino acid sequences of the single-domain antibodies are respectively shown in SEQ ID NO: 1-5; or the amino acid sequence of the single domain antibody is identical to the amino acid sequence of SEQ ID NO:1-5, 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 anti-CD 155 single domain antibody of any one of claims 1-4.

6. A recombinant protein comprising the anti-CD 155 single domain antibody of any one of claims 1 to 4.

7. A nucleotide molecule encoding the anti-CD 155 single domain antibody of any one of claims 1 to 4, characterized in that: the nucleotide sequences of the nucleotide sequences are respectively shown in SEQ ID NO:6-10, 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: 6-10, and the amino acid sequence encoded by any one of the above-mentioned amino acid sequences is identical.

8. An expression vector, characterized in that: comprising a nucleotide molecule encoding the anti-CD 155 single domain antibody 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 CD155 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 comprises a single domain antibody selected from the group consisting of anti-CD 155 according to any one of claims 1 to 4, and a pharmaceutically acceptable carrier.

11. A medicament for treating a disease characterized by: comprising as active ingredient the anti-CD 155 single domain antibody according to any one of claims 1 to 4.

12. Use of an anti-CD 155 single domain antibody according to any one of claims 1 to 4 or a 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: the diseases include solid tumor, bladder cancer, brain tumor, head and neck cancer, breast cancer, rectal cancer, prostate cancer, glioblastoma, melanoma, head and neck squamous cell carcinoma, diabetic nephropathy, diabetic retinopathy and nonalcoholic steatohepatitis.