CN116023490B - Antigen binding fragments and single chain antibodies targeting CD70 and uses thereof - Google Patents

Abstract

The invention belongs to the technical field of cellular immune engineering, and particularly relates to a fully human antigen binding fragment targeting CD70, a single-chain antibody and application thereof. The antigen binding fragment and the light chain variable region of the single chain antibody have amino acid sequences as shown in any one of Sequence No. 1-7; the amino acid Sequence of the heavy chain variable region is shown in any one of Sequence No. 8-10. The single-chain antibody provided by the invention has good affinity performance, can be combined with CD70 positive cells in flow cell detection, can be specifically combined with CD70 antigen in molecular interaction research, is a natural fully-humanized antibody source, has a sequence fully derived from a human antibody gene library, greatly reduces immunogenicity compared with a murine antibody, a chimeric antibody and a humanized antibody, and can maximally ensure safety in clinical application.

Description

Antigen binding fragments and single chain antibodies targeting CD70 and uses thereof

Technical Field

The invention belongs to the technical field of cellular immune engineering, and particularly relates to a fully human antigen binding fragment targeting CD70, a single-chain antibody and application thereof.

Background

Clinically, conventional methods for treating tumors, such as surgery, radiotherapy, chemotherapy and the like, have the bottlenecks of poor specificity, large harm to normal tissues, recurrent risks and the like, and the limitations promote the appearance of new treatment means, so that tumor immunotherapy gradually stands out due to the advantages of safety, effectiveness, low adverse reaction and the like, and becomes a fourth tumor treatment means besides surgery, radiotherapy and chemotherapy.

CD70, also known as CD27 Ligand, TNFSF7, is a type II transmembrane glycoprotein belonging to the tumor necrosis factor superfamily. In normal human tissues, CD70 is transiently expressed on activated T cells, B cells and mature Dendritic Cells (DCs), and promotes activation proliferation, functional maturation and memory cell formation of T cells by binding to CD27 protein and transmitting activation signals to the cells. Under pathological conditions, CD70 can be highly expressed in various blood and solid tumors, and currently, immunotherapeutic drugs with CD70 as a target point are applied in clinical researches for treating Acute Myeloid Leukemia (AML), renal cell carcinoma and the like.

The first generation monoclonal antibody used in treatment is murine monoclonal antibody, but the monoclonal antibody can cause immune response in human body, can be quickly cleared in the body, and brings certain difficulty to clinical application.

Disclosure of Invention

The invention aims to provide an antigen binding fragment targeting CD70, wherein the variable region of the antigen binding fragment is a natural fully-humanized antibody source, so that the immunogenicity is reduced, and the safety in clinical application is improved.

The antigen binding fragment comprises a heavy chain variable region and a light chain variable region; the light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3, and the heavy chain variable region comprises H-CDR1, H-CDR2 and H-CDR3;

further, the L-CDR1, L-CDR2 and L-CDR3 are selected from one of the following amino acid sequence combinations:

1)QSISSY、AAS、QQSYSTPWT；

2)QSVSSN、GAS、QQYNNWPLT；

3)QSISSS、AAS、QQSYNTPRT；

4)QSLLSSADDLNY、WAS、QQYYGTPT；

5)QSVSSY、GAS、QQSYTLPLT；

6)QTINTY、AAS、QQSYSTLIT；

7)QSVSSY、GAS、QQSYTLPRT；

the H-CDR1, H-CDR2 and H-CDR3 are selected from one of the following amino acid sequence combinations:

1)GYTFTDYY、INPYNGGT、ARSVYDYPFDY；

2)GDSVSSNSAA、TYYRSKWYN、ARWGPAADGGFDP

further, the amino acid Sequence of the light chain variable region of the antigen binding fragment is as shown in Sequence No.1, sequence No.2, sequence No.3, sequence No.4, sequence No.5, sequence No.6 or Sequence No.7 or a functional variant thereof; the amino acid Sequence of the heavy chain variable region is as shown in Sequence No.8, sequence No.9 or Sequence No.10 or a functional variant thereof.

Further, there is provided a full length antibody targeting CD70, said full length antibody comprising the antigen binding fragment of the foregoing.

The invention also aims to provide a single-chain antibody targeting CD70, a single-chain antibody (ScFv) drug targeting tumor has wide application prospect, and the ScFv has the following advantages as a targeting molecule: the molecular weight is small, the penetrability is strong, the humanization can be realized, the modification is easy, the large-scale production and the like can be realized, and a plurality of ScFv medicaments are available at home and abroad and are used clinically at present. The single chain antibody variable region is a natural fully human antibody source, the sequence is completely from a human antibody gene library, and compared with a murine antibody, a chimeric antibody and a humanized antibody, the immunogenicity of the single chain antibody variable region is greatly reduced, and the problem of safety in clinical application is solved.

The single chain antibody comprises a heavy chain variable region and a light chain variable region; the light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3, and the heavy chain variable region comprises H-CDR1, H-CDR2 and H-CDR3;

the L-CDR1, L-CDR2 and L-CDR3 are selected from one of the following amino acid sequence combinations:

1)QSISSY、AAS、QQSYSTPWT；

2)QSVSSN、GAS、QQYNNWPLT；

3)QSISSS、AAS、QQSYNTPRT；

4)QSLLSSADDLNY、WAS、QQYYGTPT；

5)QSVSSY、GAS、QQSYTLPLT；

6)QTINTY、AAS、QQSYSTLIT；

7)QSVSSY、GAS、QQSYTLPRT；

the H-CDR1, H-CDR2 and H-CDR3 are selected from one of the following amino acid sequence combinations:

1)GYTFTDYY、INPYNGGT、ARSVYDYPFDY；

2)GDSVSSNSAA、TYYRSKWYN、ARWGPAADGGFDP。

further, the amino acid Sequence of the light chain variable region is as shown in Sequence No.1, sequence No.2, sequence No.3, sequence No.4, sequence No.5, sequence No.6 or Sequence No.7 or a functional variant thereof; the amino acid Sequence of the heavy chain variable region is as shown in Sequence No.8, sequence No.9 or Sequence No.10 or a functional variant thereof.

Further, the light chain variable region and the heavy chain variable region are linked by a linker which may be selected from any polypeptide which may have a linking effect, preferably a polypeptide having an amino acid Sequence as shown in Sequence No.11 or a functional variant thereof, and a nucleic acid Sequence encoding the polypeptide as shown in Sequence No. 22.

Further, the amino acid sequence of the single chain antibody is preferably one comprising the following sets of light chain and heavy chain combinations:

1) The amino acid Sequence of the light chain variable region is shown as Sequence No.1, and the amino acid Sequence of the heavy chain variable region is shown as Sequence No. 8;

2) The amino acid Sequence of the light chain variable region is shown as Sequence No.2, and the amino acid Sequence of the heavy chain variable region is shown as Sequence No. 8;

3) The amino acid Sequence of the light chain variable region is shown as Sequence No.3, and the amino acid Sequence of the heavy chain variable region is shown as Sequence No. 8;

4) 3) the amino acid Sequence of the light chain variable region is shown as Sequence No.4 and the amino acid Sequence of the heavy chain variable region is shown as Sequence No. 9;

5) The amino acid Sequence of the light chain variable region is shown as Sequence No.5, and the amino acid Sequence of the heavy chain variable region is shown as Sequence No. 8;

6) The amino acid Sequence of the light chain variable region is shown as Sequence No.6, and the amino acid Sequence of the heavy chain variable region is shown as Sequence No. 10;

7) The amino acid Sequence of the light chain variable region is shown as Sequence No.7, and the amino acid Sequence of the heavy chain variable region is shown as Sequence No. 8.

Further, a CAR structure comprising the single chain antibody of any one of the preceding claims, the CAR structure further comprising a hinge region, a transmembrane region, an intracellular signaling region. The hinge region sequence may be derived from: igG, CD8, CD7, CD4 or other equivalent functional protein molecules; the transmembrane region may be derived from: CD8, CD28, CD3 epsilon, CD4, CD16, CD137, CD80, CD86 or other equivalent functional protein molecules; the intracellular signal region may be derived from: CD3, CD137, CD28, CD27, OX40, ICOS, GITR, CD2, CD40, PD-1, PD1L, B7-H3, lymphocyte function-associated antigen-1 (LFA-1), ICAM-1, CD7, NKG2C, CD, CD86 and CD 127.

Further, the CAR structure comprises one or more components of a natural killer cell receptor (NKR), thus forming a NKR-CAR. The NKR component may be a transmembrane domain, hinge domain or cytoplasmic domain from any of the following natural killer cell receptors: killer cell immunoglobulin-like receptors (KIRs), such as KIR2DL1, KIR2DL2/L3, KIR2DL4, KIR2DL5A, KIR DL5B, KIR DS1, KIR2DS2, KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1/S1, KIR3DL2, KIR3DL3, KIR2DP1, and KIR3DP1; natural Cytotoxic Receptors (NCR), e.g., NKp30, NKp44, NKp46; a family of Signaling Lymphocyte Activation Molecules (SLAM) for immune cell receptors, e.g., CD48, CD229, 2B4, CD84, NTB-A, CRA, BLAME, and CD2F-10; fc receptors (FcR), e.g., CD16, and CD64; and Ly49 receptors, e.g., ly49A, LY C. The NKR-CAR molecule may interact with an adapter molecule or an intracellular signaling domain (e.g., DAP 12).

Further, there is provided a nucleic acid Sequence comprising a single-chain antibody of any one of the preceding claims, a nucleotide Sequence encoding the light chain as shown in Sequence No.12, sequence No.13, sequence No.14, sequence No.15, sequence No.16, sequence No.17 or Sequence No. 18; the nucleotide sequences encoding the heavy chains are shown as Sequence No.19, sequence No.20, sequence No. 21.

Further, a recombinant plasmid comprising the nucleic acid sequence is provided, and the recombinant plasmid further comprises an expression vector. The expression vector is any one of a lentivirus expression vector, a retrovirus expression vector, an adenovirus expression vector, an adeno-associated virus expression vector, a DNA vector, an RNA vector and a plasmid.

In certain embodiments, the lentiviral vector is selected from the group consisting essentially of: human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), visna-maedi virus (VMV), caprine arthritis-encephalitis virus (CAEV), equine Infectious Anemia Virus (EIAV), feline Immunodeficiency Virus (FIV), bovine Immunodeficiency Virus (BIV), and Simian Immunodeficiency Virus (SIV).

In certain embodiments, the vector comprises a left (5 ') retroviral LTR, a Psi (ψ) packaging signal, a central polypurine tract/DNA FLAP (cPPT/FLAP), a retroviral export element, a promoter operably linked to a polynucleotide encoding a CAR encompassed herein, and a right (3') retroviral LTR.

In certain embodiments, the CAR comprises a hepatitis b virus posttranscriptional regulatory element (HPRE) or a Woodchuck Posttranscriptional Regulatory Element (WPRE) and an optimized woodchuck posttranscriptional regulatory element (oPRE).

In certain embodiments, the promoter of the 5' LTR is replaced with a heterologous promoter.

In certain embodiments, the heterologous promoter is a Cytomegalovirus (CMV) promoter, a rous sarcoma virus (Rous Sarcoma Virus, RSV) promoter, or a simian virus 40 (SV 40) promoter.

In certain embodiments, the 5'LTR or 3' LTR is a lentiviral LTR.

In certain embodiments, the 3' LTR is a self-inactivating (SIN) LTR.

Further, there is provided a CAR-T cell obtained by transfecting a T cell with the aforementioned expression vector.

In certain embodiments, the cells can express other active agents, e.g., an agent that enhances the activity of a CAR-expressing cell. The active agent may be an agent that blocks an inhibitory molecule. Inhibitory molecules such as PD1 may, in some embodiments, reduce the ability of CAR-expressing cells to mount an immune effector response. Inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CEACAM (CEACAM-1, CEACAM-3, CEACAM-5), LAG3, VISTA, BTLA, TIG, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD 276), B7-H4 (VTCN 1), HVEM (TNFRSF 14 or CD 270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, TGFR (TGFR beta) and TGFR beta. The extracellular domain of the inhibitory molecule may be fused to a transmembrane domain and an intracellular signaling domain, such as a PD1 CAR.

Further, the application of the antigen binding fragment, the full-length antibody, the single-chain antibody, the CAR structure, the nucleic acid sequence, the expression vector or the CAR-T cell in preparing antitumor drugs is provided. In particular, the prepared antitumor drug can be applied to the immunotherapy of blood or solid tumors aiming at CD70 targets.

Specifically, according to literature and prior art review, the types of CD70 expressing tumors are shown in table 1 below.

TABLE 1 tumor types expressing CD70

Preferably, the tumor comprises a plurality of tumor tissues such as renal cancer, hematological malignancy, thymus tumor, ovarian cancer, glioblastoma, nasopharyngeal carcinoma, and the like. More preferably, the tumor is acute myeloid leukemia or renal cell carcinoma.

Further, the application of the antigen binding fragment or the full-length antibody or the single-chain antibody in preparing a detection reagent/detection kit is provided, and the detection reagent or the detection kit can efficiently and accurately recognize the CD70 antigen.

The invention also provides a preparation method of the single-chain antibody, which is to construct a fully human single-chain antibody library by a phage display technology, and can obtain the therapeutic fully human single-chain antibody with low immunogenicity in a shorter time compared with the conventional method for obtaining the ScFv by the phage display technology.

There are four recombinant antibody platforms currently used to generate human antibodies for therapy: (1) "humanization" of mouse monoclonal antibodies; (2) immunization of a transgenic mouse containing a human antibody gene; (3) In vitro screening of fully human antibodies from a large repertoire of human antibodies; (4) technique for preparing single B cell antibody. Phage display technology is the most developed technology for preparing monoclonal antibodies. It was proposed by George Smith, university of Missouri, USA in 1985, the basic principle of which was to insert foreign DNA into the genomic DNA of a filamentous phage, fusion-express it with the coat protein of the phage, and display it on the phage surface. Through affinity panning by a proper method, phage carrying a certain fragment with specific affinity are enriched, and the gene sequence is obtained through sequencing. In certain embodiments, to screen for human antibodies targeting CD70 antigen, a repertoire of fully human single chain antibodies is constructed, normal PBMC are used as a starting material, total RNA is extracted for reverse transcription of cDNA, antibody variable regions are amplified, heavy chain variable region VH and light chain variable region VL are linked by a Linker to obtain antibody sequences, which are constructed into phagemid vectors, electrotransferred to display strains, the repertoire is constructed, the library is panned by CD70 extracellular domain antigen, and the screened clones are identified, ultimately obtaining a plurality of ScFv with specific binding to CD70 antigen.

In the present invention, the term "functional variant" is generally meant to include an amino acid sequence that has substantially the same function as it (e.g., may possess the properties of the chimeric antigen receptor) and has at least 85% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity thereto. In certain embodiments, the variant of the amino acid sequence has substantially the same function as it.

The invention has the beneficial effects that

The single chain antibody variable region provided by the invention is a natural fully human antibody source, the sequence is fully from a human antibody gene library, and compared with a murine antibody, a chimeric antibody and a humanized antibody, the immunogenicity of the single chain antibody variable region is greatly reduced, and the safety can be ensured to the greatest extent in clinical application.

The single-chain antibody provided by the invention can specifically identify the human CD70 antigen, and can be applied to the immunotherapy of blood or solid tumors aiming at a CD70 target point.

The single-chain antibody provided by the invention has good affinity performance, can be combined with CD70 positive cells in flow cytometry detection, can be combined with CD70 antigen specificity in molecular interaction research, and has potential clinical diagnosis and treatment application.

Drawings

FIG. 1 shows ELISA for positive clones.

FIG. 2 is a flow staining of K562 cells by humanized ScFv A-7-18.

FIG. 3 is a flow staining of K562-CD70 cells by humanized ScFv A-7-18.

FIG. 4 shows DK-10-A01 flow staining of K562 cells.

FIG. 5 shows DK-10-A02 flow staining of K562 cells.

FIG. 6 shows DK-10-A03 flow staining of K562 cells.

FIG. 7 shows DK-10-A04 flow staining of K562 cells.

FIG. 8 shows DK-10-A05 flow staining of K562 cells.

FIG. 9 is a flow chart of DK-10-A06 on K562 cells.

FIG. 10 is a flow chart of DK-10-A07 versus K562 cells.

FIG. 11 shows DK-10-A01 flow staining of K562-CD70 cells.

FIG. 12 shows DK-10-A02 flow staining of K562-CD70 cells.

FIG. 13 shows DK-10-A03 flow staining of K562-CD70 cells.

FIG. 14 shows the kinetics of DK-10-A04 binding to CD70 antigen.

FIG. 15 shows DK-10-A05 flow staining of K562-CD70 cells.

FIG. 16 is a flow chart of DK-10-A06 on K562-CD70 cells.

FIG. 17 is a flow chart of DK-10-A07 versus K562-CD70 cells.

FIG. 18 shows the kinetics of binding of humanized ScFv A-7-18 to CD70 antigen.

FIG. 19 shows the kinetics of DK-10-A01 binding to CD70 antigen.

FIG. 20 shows the kinetics of DK-10-A02 binding to CD70 antigen.

FIG. 21 shows the kinetics of DK-10-A03 binding to CD70 antigen.

FIG. 22 shows the kinetics of DK-10-A04 binding to CD70 antigen.

FIG. 23 shows the kinetics of DK-10-A05 binding to CD70 antigen.

FIG. 24 shows the kinetics of DK-10-A07 binding to CD70 antigen.

Detailed Description

The examples are presented for better illustration of the invention, but the invention is not limited to the examples. Those skilled in the art will appreciate that various modifications and adaptations of the embodiments described above are possible in light of the above teachings and are intended to be within the scope of the invention.

In the embodiment of the invention, the antigen panning is specifically implemented as follows:

(1) Closing: respectively re-suspending phage and CD70-protein G coupled magnetic beads by using PBS to dissolve 5% skimmed milk powder sealing solution, and uniformly mixing;

(2) Co-incubation: placing the CD70-protein G coupled magnetic beads on a magnetic rack, discarding the supernatant, and then re-suspending the magnetic beads by using phage;

(3) Cleaning: placing the magnetic bead phage mixed solution in a magnetic rack, removing the supernatant, and adding a proper volume of Tween-80 PBST cleaning solution to clean the magnetic beads;

(4) Eluting: placing the cleaned magnetic beads on a magnetic frame, drying the supernatant, adding pH2.2Gly-HCl, uniformly mixing, incubating at room temperature for 7min, adding Tris-HCl to adjust the pH to be close to neutral, finally placing the mixture on the magnetic frame, and transferring the phage supernatant to a new 1.5mL EP tube to complete one round of panning;

(5) Enrichment: inoculating phage into TGI bacterial liquid for infection, standing at 37 ℃, centrifuging, reserving 200 mu L of culture medium for resuspension precipitation, coating on a 2YTAG plate, and culturing upside down overnight;

(6) Washing the plate: the overnight cultured plates were washed from the medium to remove plaque and used as seed stock for the next round of library packaging.

In the embodiment of the invention, the specific implementation process of ELISA detection is as follows:

(1) Coating: diluting CD70 antigen with carbonate coating buffer to 1 μg/mL, adding 100 μl/well to 96-well plate, capping at 4 ℃ overnight;

(2) Obtaining phage samples: centrifuging the phage monoclonal recombinant bacteria liquid cultured overnight at 8000rpm at 4 ℃ for 10min, and taking the supernatant as a detection sample;

(3) Closing: washing the antigen coated plate on a plate washer with PBS for 3 times, and adding 5% skimmed milk powder sealing liquid into each hole for sealing;

(4) Antibody incubation: after the plate washer PBS washes the plate for 3 times, 100 mu L of phage monoclonal recombinant bacteria liquid to be detected is added into each hole, and the plate washer is incubated for 1h at 37 ℃;

(5) Adding a secondary antibody: plate washer PBS was washed 3 times with 1:5000Anti-M13-HRP secondary antibody 100 μL,37 ℃ incubation for 1h;

(6) Color development: washing the plate for 6 times by a plate washing machine, adding 100 mu L of TMB color development liquid into each hole, and standing at room temperature in a dark place for 25min for color development;

(7) And (3) terminating: 50 mu L of 2M H are added to each well ₂ SO ₄ Terminating the reaction;

(8) And (3) detection: placing the detection plate in an enzyme labeling instrument to detect OD ₄₅₀ The absorbance was 2.5 times higher than that of the negative control, and the phage clone was positive.

EXAMPLE 1 construction of fully human Single chain antibody library

PBMC separation is carried out by adopting Ficoll separating liquid, and the Ficoll separating liquid is slowly added into normal human blood so that a clear separation interface between the Ficoll separating liquid and the normal human blood is maintained. The 50mL centrifuge tube containing the blood and the separation solution was centrifuged at about 15℃for 20min. After centrifugation, the whole liquid level is divided into four layers, wherein the upper layer is a plasma mixture, the lower layer is red blood cells and granulocytes, the middle layer is Ficoll liquid, and the junction of the upper layer and the middle layer is provided with a white cloud layer narrow band mainly comprising PBMC, namely a PBMC cell layer. The upper plasma mixture was carefully aspirated with a sterile pasteur pipette, and then PBMCs were aspirated with a new sterile pasteur pipette to obtain isolated PBMCs.

Total RNA was extracted by conventional methods and reverse transcribed into cDNA. Primer design was then performed according to the method described in Sblattero, d., bradburley, a, (1998) A definitive set of oligonucleotide primers for amplifying human V regions, immunotechnology 3,271-278, in which VL was before or after, VH was after or before, and the flexible Linker was used to link the VH, the heavy and light chain variable region gene fragments of the antibodies were obtained by PCR, and ScFv nucleic acid fragments were obtained by amplification using conventional overlap PCR (PCR methods refer to "molecular cloning guidelines (Molecular Cloning: A Laboratory Manual)" (third edition), U.S. Joe Sambrook, david russell, science publishers), the ScFv nucleic acid fragments were linked to phagemid vector pComb3xss, and the products were transformed into TGI strain by electrotransfer to obtain a fully human single chain antibody library.

EXAMPLE 2 phage display of fully human Single chain antibody library preparation

Adding the library bacterial liquid into a fresh LB liquid culture medium for resuscitating culture, adding VSCM13 helper phage according to the infection complex number of VCSM 13:bacteria=50:1, fully mixing, and standing for continuous culture. The culture was centrifuged to discard the supernatant, and the pellet was resuspended in ampicillin-kanamycin-resistant SOB medium and cultured overnight. The bacterial solution was centrifuged, the supernatant was collected and 1/5 volume of NaCl solution was added, incubated on ice for 1 hour, centrifuged again, and the precipitated phage was resuspended in PBS and filtered with a filter.

Example 3 antigen panning

Incubating CD70 protein with Fc label with proteoG magnetic beads to prepare CD 70-proteoG coupled magnetic beads, pumping the coupled magnetic beads into the prepared fully human single chain antibody library phage panning, and performing 3-4 rounds of co-incubation, washing and eluting panning process to obtain the specific monoclonal antibody against antigen.

EXAMPLE 4 screening of Positive clones

After panning, the monoclonal plaques finally taken out of the warehouse are picked for ELISA detection and screening, the detection result is shown in figure 1, phage clones which are combined with the CD70 antigen are obtained, and the following 7 ScFv strains are obtained through panning, and all the monoclonal plaques have the specific binding capacity of the human CD70 antigen.

DK-10-A01 ScFv is: the amino acid Sequence of the light chain variable region is shown as Sequence No.1, the amino acid Sequence of the heavy chain variable region is shown as Sequence No.8, and the amino acid Sequence of the linker is shown as Sequence No. 11;

DK-10-A02 ScFv is: the amino acid Sequence of the light chain variable region is shown as Sequence No.2, the amino acid Sequence of the heavy chain variable region is shown as Sequence No.8, and the amino acid Sequence of the linker is shown as Sequence No. 11;

DK-10-A03 ScFv is: the amino acid Sequence of the light chain variable region is shown as Sequence No.3, and the amino acid Sequence of the heavy chain variable region is shown as Sequence No. 8; the amino acid Sequence of linker is shown in Sequence No. 11;

DK-10-A04 ScFv is: the amino acid Sequence of the light chain variable region is shown as Sequence No.4, and the amino acid Sequence of the heavy chain variable region is shown as Sequence No. 9; the amino acid Sequence of linker is shown in Sequence No. 11;

DK-10-A05 ScFv is: the amino acid Sequence of the light chain variable region is shown as Sequence No.5, and the amino acid Sequence of the heavy chain variable region is shown as Sequence No. 8; the amino acid Sequence of linker is shown in Sequence No. 11;

DK-10-A06 ScFv is: the amino acid Sequence of the light chain variable region is shown as Sequence No.6, and the amino acid Sequence of the heavy chain variable region is shown as Sequence No. 10; the amino acid Sequence of linker is shown in Sequence No. 11;

DK-10-A07 ScFv is: the amino acid Sequence of the light chain variable region is shown as Sequence No.7, and the amino acid Sequence of the heavy chain variable region is shown as Sequence No. 8; the amino acid Sequence of linker is shown in Sequence No. 11.

Example 5 expression and purification of ScFv

100ng of positive phage cloned pComb3xss plasmid was mixed with 100. Mu.L of competent Rosetta gami (DE 3) bacteria and ice-bathed for 5min, heat-shocked at 42℃for 90s, ice-bathed for 3min, and then spread onto LB plates containing ampicillin resistance, and placed in a constant temperature incubator at 37℃for overnight culture; the formed monoclonal bacterial plaque is selected and placed in 4mL of LB culture medium, after shaking culture is carried out for 8 hours at 37 ℃, bacterial liquid is transferred into 200mL of LB culture medium for culture, when the OD of the bacterial liquid reaches 0.5-1.0, IPTG is added, the final concentration is adjusted to 1mM, and induced expression is carried out for 12 hours at 37 ℃. And collecting the thalli by centrifugation at 3600rpm, adding PBS to resuspend the precipitate, carrying out ultrasonic crushing for 2min, centrifuging the lysate at 12000rpm and 4 ℃, discarding the precipitate, and collecting the supernatant for protein purification.

Filtering bacterial solution splitting supernatant with 0.22 mu m, diluting with equal volume of PBS with pH7.2, enriching ScFv with GE Ni Sepharose excel purification column, washing with 5 times of PBS with 30mM imidazole PBS solution to remove impurities, eluting protein with 500mM imidazole PBS solution, collecting washing solution, concentrating with 3kDa ultrafiltration tube, loading GE HiLoad Superdex/600 200pg molecular exclusion chromatography column, washing with PBS, collecting ultraviolet absorption peak, and performing flow-type dyeing specificity detection after SDS-PAGE identifies ScFv purification effect.

Example 6 flow-through dyeing Capacity identification

The K562 and K562-CD70 cells were individually dispensed into 1.5mL Ep tubes, each of which was 1X 10 in volume ⁶ The individual cells were all centrifuged at 1000g for 5min and the supernatant was discarded, resuspended in 50. Mu.L of 30. Mu.g/mL A-7-18 (humanized anti-human CD70 ScFv (antibody of another unpublished patent 202010559366.8 of the applicant), the experimental positive control)/DK-10-A01/DK-10-A02/DK-10-A03/DK-10-A04/DK-10-A05/DK-10-A06/DK-10-A07 ScFv solution and incubated at 4℃in the absence of light. After 30min, PBS was added to resuspend the cells, 1000g was washed for 5min, the supernatant was discarded, 30. Mu.L of Anti-His-647 fluorescent secondary antibody for detection was added to resuspend the cells, and after 4℃incubation was performed in the dark for 30min; cells were resuspended in 1mL of PBS and washed twice in 1000g of 5min, and the supernatant was discarded and resuspended in 200. Mu.L of PBS. All cell tubes were run on-machine to detect the positive rate of flow staining, and the staining results of the anti-CD 70 ScFv staining group were compared with those of the A-7-18 staining group to evaluate the flow staining specificity of ScFv. The result of staining K562 with A-7-18ScFv is shown in FIG. 2, the result of staining K562-CD70 cells with A-7-18ScFv is shown in FIG. 3, and the result of staining K562 cells with CD 70-specific ScFv obtained by panning is shown in FIG. 4, FIG. 5, FIG. 6, FIG. 7, FIG. 8, FIG. 9 and FIG. 10, and it is seen from the figure that CD 70-specific ScFv is not bound to negative cells; the staining results of K562-CD70 are shown in FIG. 11, FIG. 12, FIG. 13, FIG. 14, FIG. 15, FIG. 16 and FIG. 17, and the binding of CD 70-specific ScFv to positive cells is shown in the figures, which proves that the screened CD 70-specific ScFv can be used as a detection reagent for detecting the expression of CD 70.

EXAMPLE 7 kinetic parameter detection of antigen-antibody binding

The ProA biosensor was immobilized with ligand protein in 30ug/mL of CD70-hFc solution loading 120s in the assay buffer, loading 120s in the gradient diluted analyte (CD 70 ScFv) solution association 120s, and finally, in the assay buffer association 300s. Whether specific binding exists or not is judged by changing the response value R, and in general, the specific binding can be realized when the maximum response value Rmax after the Reference is leveled is larger than 0.05nm and the concentration dependence and dissociation phenomenon exists. The kinetic parameters represented by the binding-dissociation curves, such as binding constant (Kon), dissociation constant (Kdis) and equilibrium dissociation constant (KD), can be given by the Data Analysis software carried by the Fortebio oct K2 instrument. Wherein the unit of Kon is 1/Ms to express the rate of binding of antigen to antibody, the higher the Kon, the faster the antibody binds to antigen to form a complex; the Kdis unit is 1/s, which is used for expressing the dissociation rate of antigen and antibody, and the higher the Kdis, the faster the dissociation rate of the antigen-antibody complex; KD is the ratio of Kdis to Kon to comprehensively describe how easily an antigen-antibody binds, the smaller the KD, the higher the antibody affinity is generally considered. The binding kinetics data of A-7-18ScFv and CD70 antigen are shown in Table 1, the graph is shown in FIG. 18, the binding kinetics data of panning to obtain CD 70-specific ScFv and CD70 antigen are shown in tables 2-7, and the graphs are shown in FIG. 19, FIG. 20, FIG. 21, FIG. 22, FIG. 23 and FIG. 24, respectively.

TABLE 1 kinetic parameters of the binding of humanized ScFv A-7-18 to CD70 antigen

TABLE 2 kinetics of binding of DK-10-A01 to CD70 antigen

Table 3 DK-10-A02 binding kinetics parameters to CD70 antigen

TABLE 4 kinetic parameters of binding of DK-10-A03 to CD70 antigen

TABLE 5 kinetics of binding of DK-10-A04 to CD70 antigen

TABLE 6 kinetics of binding of DK-10-A05 to CD70 antigen

TABLE 7 kinetics of binding of DK-10-A07 to CD70 antigen

Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.

Sequence listing

<110> Chongqing precision biotechnology Co., ltd., chongqing precision biotechnology industry research institute Co., ltd

<120> antigen binding fragments and single chain antibodies targeting CD70 and uses thereof

<130> 2021-9-1

<160> 22

<170> SIPOSequenceListing 1.0

<210> 1

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 1

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Phe Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Asp Ile Lys

100 105

<210> 2

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 2

Glu Thr Thr Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 3

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 3

Ala Ile Arg Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Ser

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Val Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Asn Thr Pro Arg

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys

100 105

<210> 4

<211> 112

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 4

Asp Ile Gln Met Thr Gln Ser Pro Asp Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Ser Ser

20 25 30

Ala Asp Asp Leu Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Gln Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Asn Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln

85 90 95

Tyr Tyr Gly Thr Pro Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105 110

<210> 5

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 5

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Tyr Thr Leu Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 6

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 6

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Thr Ile Asn Thr Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Leu Ile

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 7

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 7

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Phe Gly Ala Ser Ser Leu Ala Thr Gly Val Pro Tyr Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Leu Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Asp Ile Lys

100 105

<210> 8

<211> 118

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 8

Glu Val Gln Leu Val Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Met His Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Tyr Asn Gly Gly Thr Asp Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Val Tyr Asp Tyr Pro Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 9

<211> 118

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 9

Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Met His Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Tyr Asn Gly Gly Thr Asp Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Val Tyr Asp Tyr Pro Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 10

<211> 123

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 10

Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn

20 25 30

Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala

50 55 60

Val Ser Val Lys Asp Arg Val Thr Ile Asn Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Gln Leu Arg Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Arg Trp Gly Pro Ala Ala Asp Gly Gly Phe Asp Pro

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 11

<211> 19

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 11

Arg Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser

1 5 10 15

Thr Lys Gly

<210> 12

<211> 321

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctttgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta ccccctggac gttcggccaa 300

gggaccaaac tggatatcaa a 321

<210> 13

<211> 321

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

gaaacgacac tcacgcagtc tccagccacc ctgtctgtgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtgttagc agcaacttag cctggtacca gcagaaacct 120

ggccaggctc ccaggctcct catctatggt gcatccacca gggccactgg tatcccagcc 180

aggttcagtg gcagtgggtc tgggacagag ttcactctca ccatcagcag cctgcagtct 240

gaagattttg cagtttatta ctgtcagcag tataataact ggcccctcac tttcggcgga 300

gggaccaagg tggagatcaa a 321

<210> 14

<211> 321

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

gccatccgga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc agctctttaa attggtatca gcagaaacca 120

gggaaagccc ctaagcttgt gatctatgct gcgtccagtt tacaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacttacta ctgtcaacag agttacaata ccccgagaac tttcggccct 300

gggaccaagg tggagatcaa a 321

<210> 15

<211> 336

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

gacatccaga tgacccagtc tccagactcc ctgcctgtgt ctctgggcga gagggccacc 60

atcaattgca agtccagcca gagtcttcta tccagcgccg acgatttgaa ctacttagct 120

tggtaccagc agaaaccagg gcagcctcct aagctgctca tttactgggc atctacccgg 180

caatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240

atcaacagcc tgcaggctga agatgtggca gtttatttct gtcagcaata ttatggtact 300

cccaccttcg gccaagggac acgactggag attaaa 336

<210> 16

<211> 321

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

gaaattgtgt tgacgcagtc tccagccacc ctgtctttgt ctccagggga gagagccacc 60

ctctcctgca gggccagtca gagtgttagc agctacttag cctggtacca acagaaacct 120

ggccaggctc ccaggctcct catctatggt gcatccagca gggccactgg catcccagac 180

aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag actggagcct 240

gaagattttg cagtgtatta ctgtcaacag agttacactc ttcctctgac attcggccag 300

gggaccaagc tggagatcaa a 321

<210> 17

<211> 321

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

gacatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gaccattaac acctatttaa attggtatca acagaaacca 120

gggaaagccc ctaagcccct gatttatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat tttactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta ccctgatcac cttcggccaa 300

gggacacgac tggagattaa a 321

<210> 18

<211> 321

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

gacattgtga tgacccagtc tccatcctcc ctgtctttgt ctccaggaga gagagccacc 60

ctctcctgcc gggccagtca gagtgttagc agctacttag cctggtacca acagaaacct 120

gggaaagccc ccaagctcct catctttggt gcatccagtt tggccactgg ggtcccatac 180

aggttcagtg gcagtggatc tgggacagac ttcactctca ccatcagcag actgcagcct 240

gaagattttg caacgtatta ctgtcaacag agttacactc ttcctcggac attcggccaa 300

gggaccaaac tggatatcaa a 321

<210> 19

<211> 354

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

gaggtgcagc tggtgcagtc cggaccagag gtgaagaagc caggagccag cgtgaaggtg 60

tcctgtaagg cctctggcta caccttcaca gattactata tgaactgggt gcggcagatg 120

cacggcaagg gactggagtg gatgggcgtg atcaacccat acaatggcgg caccgattat 180

aatcagaagt ttaagggcag agtgaccatc acagccgaca agtccacctc tacagcctac 240

atggagctga gctccctgag gagcgaggac acagccgtgt actattgtgc ccgctccgtg 300

tacgactatc cctttgatta ttggggccag ggcaccctgg tcacagtctc ctca 354

<210> 20

<211> 354

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

caggtacagc tgcagcagtc cggaccagag gtgaagaagc caggagccag cgtgaaggtg 60

tcctgtaagg cctctggcta caccttcaca gattactata tgaactgggt gcggcagatg 120

cacggcaagg gactggagtg gatgggcgtg atcaacccat acaatggcgg caccgattat 180

aatcagaagt ttaagggcag agtgaccatc acagccgaca agtccacctc tacagcctac 240

atggagctga gctccctgag gagcgaggac acagccgtgt actattgtgc ccgctccgtg 300

tacgactatc cctttgatta ttggggccag ggaaccctgg tcaccgtctc ctca 354

<210> 21

<211> 369

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

caggtacagc tgcagcagtc aggtccagga ctggtgaagc cctcgcagac cctctcactc 60

acctgtgcca tctccgggga cagtgtctct agcaacagtg ctgcttggaa ctggatcagg 120

cagtccccat cgagaggcct tgagtggctg ggaaggacat attacaggtc caagtggtat 180

aatgattatg cagtatctgt gaaagatcga gtcaccatca acccagacac ctcgaagaac 240

cagttctccc tgcagctaag gtctgtgact cccgaggaca cggctgtcta ttactgtgca 300

cgatgggggc cagcagcgga tggcgggttc gacccctggg gccagggaac cctggtcaca 360

gtctcctca 369

<210> 22

<211> 57

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 22

cgtggcagca caagcggaag cggcaaacca ggaagcggag aaggaagcac caaggga 57

Claims (8)

1. An antigen-binding fragment targeting CD70, wherein said antigen-binding fragment comprises a heavy chain variable region and a light chain variable region; the light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3, and the heavy chain variable region comprises H-CDR1, H-CDR2 and H-CDR3;

the amino acid sequence of the L-CDR1 is QSISY, the amino acid sequence of the L-CDR2 is AAS, and the amino acid sequence of the L-CDR3 is QQSYSTPWT; the amino acid sequence of the H-CDR1 is GYTFTDYY, the amino acid sequence of the H-CDR2 is INPYNGGT, and the amino acid sequence of the H-CDR3 is ARSVYDYPFDY.

2. A full length antibody targeting CD70, wherein the full length antibody comprises the antigen binding fragment of claim 1.

3. A single chain antibody targeting CD70, characterized in that said single chain antibody comprises a heavy chain variable region and a light chain variable region; the light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3, and the heavy chain variable region comprises H-CDR1, H-CDR2 and H-CDR3;

the amino acid sequence of the L-CDR1 is QSISY, the amino acid sequence of the L-CDR2 is AAS, and the amino acid sequence of the L-CDR3 is QQSYSTPWT; the amino acid sequence of the H-CDR1 is GYTFTDYY, the amino acid sequence of the H-CDR2 is INPYNGGT, and the amino acid sequence of the H-CDR3 is ARSVYDYPFDY.

4. The single chain antibody of claim 3, wherein the amino acid sequence of the light chain variable region is as shown in sequence No. 1; the amino acid Sequence of the heavy chain variable region is shown in Sequence No. 8.

5. The single chain antibody according to claim 3 or 4, wherein the light chain variable region and the heavy chain variable region are linked by a linker having an amino acid Sequence as shown in Sequence No. 11.

6. A polynucleotide encoding the single chain antibody of any one of claims 3-5, wherein the nucleotide Sequence encoding the light chain is as set forth in Sequence No. 12; the nucleotide Sequence encoding the heavy chain is shown in Sequence No. 19.

7. A recombinant plasmid comprising the polynucleotide of claim 6.

8. Use of the antigen binding fragment of claim 1 or the full length antibody of claim 2 or the single chain antibody of any one of claims 3-5 in the preparation of a detection reagent/detection kit.