CN106674347B

CN106674347B - Novel auristatin antibody

Info

Publication number: CN106674347B
Application number: CN201610680614.8A
Authority: CN
Inventors: 姜静; 王凌; 李伟伟
Original assignee: Binzhou Medical College
Current assignee: Binzhou Medical College
Priority date: 2016-08-16
Filing date: 2016-08-16
Publication date: 2019-12-13
Anticipated expiration: 2036-08-16
Also published as: CN106674347A

Abstract

The invention provides a novel auristatin antibody or a functional fragment thereof, which has stronger affinity with auristatin and high sensitivity. The invention also provides a method for detecting auristatin and a coupled drug thereof by using the novel auristatin antibody or the functional fragment thereof.

Description

Novel auristatin antibody

Technical Field

The invention relates to a novel auristatin antibody or a functional fragment thereof, which comprises a heavy chain and a light chain, and also relates to a kit prepared by using the antibody, and application of the antibody in quantitative detection of auristatin and a coupling drug thereof.

Technical Field

auristatin (dolastatin), also known as dolastatin, is a linear depsipeptide toxin protein extracted from sea mollusk tail-amputated sea hare, and has various derivatives, which are commonly known as: dolastatin-10, Dolastatin-15, TZT-1027, MMAE (monomethyyl Auristatin E), MMAF (monomethyyl Auristatin F), cimadrol, tacidodine, etc. (Bai RL, Pettit GR, Hamel E.dolastatin 10, a powerfullpeptidized peptide from a animal. inhibition of hybridization polymerization mediated through the vinca alkaloid binding domain [ J ]. Biochem pharmacol, 1990,39 (12): 1-1949.). Auristatins exert potent cytotoxic activity by acting on tubulin, and a variety of dolastatin derivatives have been developed for use in drug therapy (Bai RL, Pettit GR, Hamel E.binding of dolastatin 10 toxin a dis site for peptide anti-viral agents and vinca alkaloid sites [ J ]. J biol chem,1990,265 (28): 17141-17149.).

Because auristatin has strong cytotoxicity and lower safety of direct administration, and the "therapeutic window" is narrow, one of the most important strategies is targeted drug treatment of auristatin. The auristatin is carried to a specific part of an organism to play the drug effect by preparing a preparation with a targeting function or connecting molecules with the targeting function. The most common form of targeted Drug delivery is Antibody-conjugated drugs (ADC), and there are currently several ADC drugs of auristatin, of which 1 (Adcetris, SGN-35) has been approved for clinical study (Deng CC, Pan BQ, Owen A. Brentuximab vedotin [ J ]. Clin Cancer Res, 2013, 19 (1): 22-27.).

Quantitative pharmacological research is one of important research aspects in the development of drug effect, drug generation and toxin generation research of auristatin-related targeted drugs. The establishment of a quantitative detection method for auristatin-conjugated drugs supports the above-mentioned research

Importantly, the quantitative detection methods comprise a liquid phase-mass spectrometry method (LC-MS, LC-MS/MS), an enzyme-linked immunosorbent assay (ELISA) and the like. The enzyme-linked immunosorbent assay (ELISA) is characterized in that known antigen or antibody is firstly fixed on the surface of a solid phase carrier, an object to be detected is usually equivalent to the antibody or the antigen and is specifically combined with the antigen or the antibody combined on the surface of the solid phase carrier, the object to be detected is connected with enzyme, and quantitative analysis is carried out through the color reaction between the enzyme and a substrate (the color depth after reaction is in a certain proportion relation with the concentration of the substance to be detected).

One of the major strategies for quantitative detection of auristatin and its conjugate drugs by ELISA is capture or detection using anti-drug antibodies. In the detection process, the acquisition of the anti-drug antibody with strong specificity and high affinity is the key, and most small-molecule drugs only have reactionogenicity but no immunogenicity, which brings the problem of difficult solution for ELISA quantitative detection of auristatin and its conjugate drugs.

The invention provides a technical scheme for solving the problems, prepares the antibody specifically combined with auristatin, has strong specificity, high affinity and high sensitivity, and can be used for the quantitative detection of ELISA.

Disclosure of Invention

In order to solve the problems, the invention provides a novel auristatin antibody or a functional fragment thereof, which is an antibody with high affinity with auristatin drug molecules, and is obtained by coupling auristatin with different carrier proteins to form an artificial antigen, carrying out animal immunization and screening. The coupling strategy is mainly characterized in that after groups of drug molecules are activated, different carrier proteins are coupled to prepare artificial antigens, one is used as an immune antigen, and the other is used for screening the antigen, so that the generated antibody is directed against auristatin molecules but not against the carrier proteins. The invention also provides a method for quantitatively detecting auristatin or a conjugate thereof by using the novel auristatin antibody or the functional fragment thereof, and a kit for preparing free auristatin or auristatin conjugate drugs by using the novel auristatin antibody or the functional fragment thereof.

The specific technical scheme of the invention is as follows:

In one aspect, the present invention provides a novel auristatin antibody or functional fragment thereof comprising a heavy chain and a light chain, in particular: (i) the heavy chain comprises at least three CDR regions, and the CDR regions have amino acid sequences shown as SEQ ID NO 1, 2, 3, 7, 8, 9, 13, 14 and 15; and/or (ii) the light chain comprises at least three CDR regions having amino acid sequences as set forth in SEQ ID NOs 4, 5, 6, 10, 11, 12, 16, 17.

specifically, the novel auristatin antibody or functional fragment thereof comprises a heavy chain and a light chain, wherein (i) the heavy chain comprises three CDR regions, and the CDR regions have amino acid sequences shown as SEQ ID NO. 1, 2 and 3; and/or (ii) the light chain comprises three CDR regions having amino acid sequences as set forth in SEQ ID NOs 4, 5, 6.

Specifically, the novel auristatin antibody or functional fragment thereof comprises a heavy chain and a light chain, wherein (i) the heavy chain comprises three CDR regions with amino acid sequences shown as SEQ ID NO. 7, 8 and 9; and/or (ii) the light chain comprises three CDR regions having amino acid sequences as set forth in SEQ ID NOs 10, 11, 12.

In particular, the novel auristatin antibodies or functional fragments thereof, which comprise a heavy chain and a light chain, in particular: (i) the heavy chain comprises three CDR regions, and the CDR regions have amino acid sequences shown as SEQ ID NO 13, 14 and 15; and/or (ii) the light chain comprises three CDR regions having amino acid sequences as set forth in SEQ ID NOs 16, 5, 17.

More specifically, the novel auristatin antibody or functional fragment thereof comprises a heavy chain and a light chain, (i) the heavy chain comprises three CDR regions, CDR1 has the amino acid sequence shown in SEQ ID No. 1, CDR2 has the amino acid sequence shown in SEQ ID No. 2, CDR3 has the amino acid sequence shown in SEQ ID No. 3; (ii) the light chain comprises three CDR regions, wherein the CDR1 has an amino acid sequence shown as SEQ ID NO. 4, the CDR2 has an amino acid sequence shown as SEQ ID NO. 5, and the CDR3 has an amino acid sequence shown as SEQ ID NO. 6.

more specifically, the novel auristatin antibody or functional fragment thereof comprises a heavy chain and a light chain, (i) the heavy chain comprises three CDR regions, CDR1 has the amino acid sequence shown in SEQ ID No. 7, CDR2 has the amino acid sequence shown in SEQ ID No.8, CDR3 has the amino acid sequence shown in SEQ ID No. 9; (ii) the light chain comprises three CDR regions, wherein the CDR1 has an amino acid sequence shown as SEQ ID NO. 10, the CDR2 has an amino acid sequence shown as SEQ ID NO. 11, and the CDR3 has an amino acid sequence shown as SEQ ID NO. 12.

more specifically, the novel auristatin antibody or functional fragment thereof comprises a heavy chain and a light chain, (i) the heavy chain comprises three CDR regions, CDR1 has the amino acid sequence shown in SEQ ID No. 13, CDR2 has the amino acid sequence shown in SEQ ID No. 14, CDR3 has the amino acid sequence shown in SEQ ID No. 15; (ii) the light chain comprises three CDR regions, wherein the CDR1 has an amino acid sequence shown as SEQ ID NO. 16, the CDR2 has an amino acid sequence shown as SEQ ID NO. 5, and the CDR3 has an amino acid sequence shown as SEQ ID NO. 17.

Further, said novel auristatin antibody or functional fragment thereof, comprising a heavy chain and a light chain, characterized in that: (i) a heavy chain variable region comprising the amino acid sequences set forth as SEQ ID NOs 18, 20, 22; (ii) a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs 19, 21, 23.

In particular, said novel auristatin antibody or functional fragment thereof, comprising a heavy chain and a light chain, characterized in that: (i) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO 18; (ii) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO 19.

In particular, said novel auristatin antibody or functional fragment thereof, comprising a heavy chain and a light chain, characterized in that: (i) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO 20; (ii) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 21.

In particular, the novel auristatin antibodies or functional fragments thereof, which comprise a heavy chain and a light chain, in particular: (i) a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 22; (ii) a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 23.

Further, the novel auristatin antibody or a functional fragment thereof, wherein the antibody is a monoclonal antibody.

Further, the novel auristatin antibody or a functional fragment thereof, wherein the functional fragment is Fv, scFv, Fab, F (ab ')2, Fab', scFv-Fc fragment or Fv-PEG, scFv-PEG, Fab-PEG.

Further, the novel Auristatin antibody or functional fragment thereof, wherein the Auristatin is Dolastatin-10, Dolastatin-15, TZT-1027, MMAE (monomethyyl Auristatin E), MMAF (monomethyyl Auristatin F), Simadudine, Taxidopidine, etc., preferably MMAE, MMAF.

further, the affinity of the antibody of the novel auristatin to MMAE is 10^-8～10^-10In the meantime.

In another aspect, the present invention provides an isolated polynucleotide capable of encoding any of the auristatin antibodies or functional fragments thereof described above.

In another aspect, the invention provides an expression vector comprising the isolated polynucleotide.

In another aspect, the invention provides a host cell comprising said expression vector.

In another aspect, the invention also provides antibody-conjugated drugs containing the novel auristatin antibody or functional fragments thereof.

On the other hand, the invention also provides a medicine, a detection reagent or a kit containing the novel auristatin antibody or the functional fragment thereof.

On the other hand, the invention also provides application of the novel auristatin antibody or the functional fragment thereof in detecting auristatin or a conjugate drug thereof, wherein the application refers to the application of the auristatin antibody in an ELISA method.

Description of the drawings:

FIG. 1 shows the specificity and sensitivity of auristatin monoclonal antibodies, which were evaluated by adding auristatin as a competitive binder while binding antigen. Wherein A is a control antibody (control), which has no specific binding with antigen, has no obvious competitive action after adding auristatin with different concentrations, and B, C, D is the competitive binding effect of 1G1, 2F10 and 4C3 monoclonal antibodies respectively.

FIG. 2 shows a standard curve of auristatin in rat serum by competitive ELISA method established with auristatin monoclonal antibody.

FIG. 3 shows a standard curve for quantitative determination of auristatin conjugate in monkey serum by ELISA method established by auristatin monoclonal antibody.

The specific implementation mode is as follows:

Defining:

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. With regard to the definitions and terminology in this field, the expert can refer in particular to Current Protocols in molecular Biology (Ausubel). The abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

Notwithstanding that the numerical ranges and parameter approximations set forth the broad scope of the invention, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective measurements. In addition, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of "1 to 10" should be considered to include any and all subranges between (and inclusive of) the minimum value of 1 and the maximum value of 10; that is, all subranges beginning with a minimum value of 1 or more, e.g., 1to 6.1, and ending with a maximum value of 10 or less, e.g., 5.5 to 10. Additionally, any reference that is said to be "incorporated herein" is to be understood as being incorporated in its entirety.

It should also be noted that, as used in this specification, the singular forms "a," "an," and "the" include plural referents unless the context clearly and clearly dictates otherwise. The term "or" may be used interchangeably with the term "and/or" unless the context clearly dictates otherwise.

As used herein, "CDR regions" or "CDRs" refer to the hypervariable regions of the heavy and light chains of an immunoglobulin, as defined by Kabat et al (Kabat et al, Sequences of proteins of immunological interest,5th ed., u.s.department of Health and Human Services, NIH,1991, and later versions). There are three heavy chain CDRs and three light chain CDRs. As used herein, the term CDR or CDRs is intended to indicate one of these regions, or several or even all of these regions, which comprise the majority of the amino acid residues responsible for binding by the affinity of the antibody for the antigen or its recognition epitope, as the case may be.

For the purposes of the present invention, "identity" or "similarity" between two nucleic acid or amino acid sequences means the percentage of identical nucleotides or identical amino acid residues between the two sequences to be compared, obtained after optimal alignment (optimal alignment), which percentage is purely statistical and the differences between the two sequences are randomly distributed and cover their full length. Sequence comparison between two nucleic acid or amino acid sequences is usually carried out by comparing these sequences after they have been matched in an optimal manner, the comparison being able to be carried out by means of segments or by means of "comparison windows". In addition to being able to be performed manually, optimal alignments for comparing sequences can be performed by the local homology algorithm of Smith and Waterman (1981) [ Ad. App. Math.2:482], by the local homology algorithm of Neddleman and Wunsch (1970) [ J.MoI.biol.48:443], by the similarity search method of Pearson and Lipman (1988) [ Proc. Natl.Acad.Sci.USA85:2444), by Computer software using these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics software Package, Genetics Computer Group,575 Science Dr., Madison, Wis, or by BLAST N or BLAST P comparison software).

The term "antibody" refers to whole antibodies and any antigen-binding fragment ("antigen-binding portion") or single chain thereof. A "full-length antibody" refers to a protein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated VH) and a heavy chain constant region. The heavy chain constant region comprises three domains (domains), CH1, CH2, and CH 3. Each light chain comprises a light chain variable region (abbreviated VL) and a light chain constant region. The light chain constant region comprises a domain, CL. The VH and VL regions can also be subdivided into regions of high variability, called Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, called Framework Regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. These variable regions of the heavy and light chains comprise binding domains that interact with antigens. The constant regions of antibodies may mediate the binding of the immunoglobulin to tissues or factors of the host, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq). Chimeric or humanized antibodies are also encompassed by the antibodies according to the invention.

The term "monoclonal antibody" refers to a preparation of antibody molecules having a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

The term "functional fragment" as used herein refers in particular to an antibody fragment such as Fv, scFv (sc refers to single chain), Fab, F (ab ')2, Fab', scFv-Fc fragment or diabody (diabody), or any fragment which should be able to increase the half-life by chemical modification, e.g. addition of a poly (alkylene) glycol such as polyethylene glycol ("pegylation, pegylation") (pegylated fragments known as Fv-PEG, scFv-PEG, Fab-PEG, F (ab ')2-PEG or Fab' -PEG) ("PEG" being polyethylene glycol), or by incorporation into liposomes, said fragment having auristatin binding activity. Preferably, said functional fragment will consist of or comprise a partial sequence of the heavy or light variable chain of the antibody from which it is derived, said partial sequence being sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived, preferably at least equal to 1/100, and in a more preferred manner at least equal to 1/10, of the affinity of the antibody from which it is derived, for auristatin E. Such functional fragments will comprise a minimum of 5 amino acids, preferably 10, 15, 25, 50 and 100 consecutive amino acids of the antibody sequence from which they are derived.

In general, for the preparation of monoclonal Antibodies or functional fragments thereof, in particular of murine origin, reference may be made to the techniques described in particular in the Manual "Antibodies" (Harlow and Lane, Antibodies: organism Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY, pp.726,1988) or to the techniques described by Kohler and Milstein, prepared from hybridoma cells (Nature,256:495-497, 1975).

The term "auristatin," also known as "Dolastatin peptide," as used herein, refers to polypeptides and derivatives thereof isolated from the marine organism dolabella auricularia, including, but not limited to Dolastatin 10(Dolastatin-10, auristatin E), Dolastatin 15(Dolastatin-15, auristatin F). Auristatin (dolastatin peptide) is a mitotic inhibitor that exhibits strong anticancer activity and is therefore a candidate as an anticancer drug. Researchers have further discovered and synthesized a number of dolastatin peptide derivatives, such as Auristatin E (MMAE) and Auristatin F (MMAF), MMAE (monomethyyl Auristatin E), MMAF (monomethyyl Auristatin F), TZT-1027, Cimadudine, Taxidopidine, and the like.

The term "conjugate Drug" as used herein in the context of "auristatin and its conjugate Drug" refers to all drugs produced by conjugation with auristatin, most commonly in the form of Antibody conjugate drugs (ADCs).

The following examples serve to demonstrate and further explain some preferred embodiments and aspects of the invention, but without limiting the scope of the invention.

Example 1 preparation and sequence analysis of anti-auristatin murine monoclonal antibodies

1) Preparation of auristatin artificial antigen

The activation of the drug molecules is carried out by the azo method or the maleimide method. Among them, the azo method can be referred to in the literature (Erlanger BF. the preparation of antigenic hapatent-carrier conjugate: a surfymethod in enzymology [ M ]]New York, Academic Press 1980: 85-103; ) Carrying out the following steps: collecting auristatin (MMAE/MMAF) 0.5%Adding 1.5ml of the stock solution into a test tube, adjusting the pH value to 2.5 by using 1mol/L hydrochloric acid, dropwise adding 0.5ml of sodium nitrite solution while shaking, incubating for 1 hour at 4 ℃ in a dark place, adding 2-3 drops of ammonia sulfamate solution, and removing free nitrite ions. Finally, the volume is measured out, and the auristatin concentration is calculated to be coupled with BSA. Adding 2% BSA aqueous solution at a ratio of 20:1 (azo-activating molecule: BSA), adjusting pH to 7.5 with sodium hydroxide and incubating at 4 ℃ for 24 hours, followed by dialysis with 0.01mol/L PBS at pH7.4 by a factor of 106, preservation at 4 ℃, KLH, immunoglobulin (IgG) coupling according to the above method; coupling with maleimides is described in the literature (Greg T. Hermanson. bioconjugate Techniques (2)^ndedition), chapter19.preparation of happten-carrier ligands, essence inc. academic press.2008:746-782) and reference to the literature (Hamblett, k.j., et al, Effects of drug loading on the antibiotic resistance. clin Cancer Res,2004.10(20): p.7063-70) for coupling of carrier proteins, 1% BSA protein solution cooled to 4 ℃, final concentration 25% DMSO, in a molar ratio of drug molecules to protein 20:1, adding MC-linker-MMAE/MMAF (0.01mol/L, dissolved in DMSO), stirring at room temperature for reaction for 1h, and dialyzing by 106 times for treatment. Storage at 4 ℃ and KLH, IgG coupling were as described above.

2) Immunization and preparation of hybridomas

The auristatin-carrier protein prepared above is used as an antigen to immunize mice to prepare monoclonal antibodies. The immune response, hybridoma cell fusion and preliminary screening were all performed according to standard procedures (reference: WHO Technical Report Series, No.822,1992 Annex 3). 6 Balb/c mice (purchased from Shanghai Slek laboratory animals, Inc.) were randomly divided into 2 groups of 3 mice each. One group was immunized with auristatin-BSA and the other group was immunized with auristatin-KLH. Respectively taking 50 mu L of immunogen with 1mg/ml for the first immunization, fully emulsifying the immunogen with equivalent Freund complete adjuvant, subcutaneously injecting the neck of a mouse, performing the 2 nd injection after 2 weeks, using Freund incomplete adjuvant (Difco Lab), wherein the antigen amount is 25-50 mu g/0.05 ml/mouse, performing the 3 rd injection after 3 weeks, the injection dose is the same as the 2 nd injection, and taking blood 10 days after the 3 rd injection. The serum of the mouse is detected by enzyme-linked immunosorbent assay (ELISA), and the detection antigen adopts auristatin to couple with artificial antigen different from immune antigen carrier protein. The spleen of the mouse with the highest titer of anti-auristatin antibodies in the serum was removed and fused with myeloma cell P3X63Ag8(ATCC CRL-1580). The fused cells were diluted in 10 96-well plates and primary screened by ELISA based on the binding ability to auristatin antibody-auristatin. In a typical ELISA experiment, Nunc Maxisorb 96-well plates were coated with auristatin-BSA/KLH (0.2-1(g/ml), and then incubated with mouse serum or hybridoma supernatant (100. mu.L) diluted in a gradient₂An anti-mouse IgG Fc-specific secondary antibody (Invitrogen) was detected.

The supernatants of 400 hybridoma cell lines were screened by ELISA, 36 of which showed strong auristatin-ECD binding. Ten hybridoma cells with the strongest auristatin binding capacity are selected, and subcloned hybridoma cell strains are screened by a limiting dilution method. The method comprises the steps of culturing a suspension subclone hybridoma cell strain, purifying protein, determining the binding affinity of auristatin by ELISA, and further testing the binding capacity of auristatin by a competitive ELISA method of auristatin molecules. Finally 3 hybridoma cell lines were determined by sequence analysis: 1G1, 2F10, 4C3, which have strong auristatin binding ability and are subsequently analyzed further by ELISA and cell assays.

3) Sequence analysis of anti-auristatin hybridoma cell clones

The sequences of the variable regions of the heavy and light chains cloned from the above hybridoma cells were obtained by rapid Amplification of the 5' ends using a commercial Kit SMARTTM RACE cDNA Amplification Kit (Clontech Co.) and sequencing. Total RNA was extracted from hybridoma cells using the RNApureTissue Kit (Biotech Co., Ltd., Kangwei, Beijing) using SMART^TMReverse transcription of total RNA is carried out by RACE cDNA Amplification Kit, total RNA is used as a template, Reverse Transcriptase SMARTScript (TM) Reverse Transcriptase is added by using a primer in the Kit, Reverse transcription is carried out according to the steps provided by the Kit to obtain RACE-Ready first strand cDNA, then two rounds of PCR are carried out, and the first round of PCR is carried out so as to obtain RACE-Ready first strand cDNAThe obtained cDNA is used as a template, and UPM provided in the kit is a 5 'end primer and a 3' end primer. The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; 25 amplification cycles (denaturation at 94 ℃ for 30s, annealing at 68 ℃ for 30s, and extension at 72 ℃ for 2 min); finally, extension is carried out for 10min at 72 ℃. And in the second round of PCR, the product of the first round of PCR is used as a template, the NUP provided in the kit is used as a 5 'end primer and a 3' end primer, and the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; 25 amplification cycles (denaturation at 94 ℃ for 30s, annealing at 68 ℃ for 30s, and extension at 72 ℃ for 2 min); extension at 72 ℃ for 10 min. The variable regions of the heavy and light chains were cloned from the hybridoma cells described above. The sequences of the three cell lines expressing the monoclonal antibodies are shown in table 1:

TABLE 1 variable region amino acid sequences of anti-auristatin monoclonal antibodies

the PCR product was purified by agarose gel electrophoresis and subcloned into pCR2.1TOPO cloning vector (Invitrogen). Plasmid DNA from independent clones was obtained by PCR and sequenced with M13 forward and reverse primers. The amino acid sequences of the Complementarity Determining Regions (CDRs) are defined by the Kabat coding table and are listed in table 2.

TABLE 2 amino acid sequences of the CDRs of anti-auristatin monoclonal antibodies

Example 2 specificity and sensitivity of Auistatin monoclonal antibodies

In order to detect the specificity and sensitivity of the auristatin monoclonal antibody, IgG protein coupled with auristatin is coated in a 96-well plate (300ng/well), monoclonal antibodies (1G1, 2F10 and 4C3) are diluted in a gradient (1:500,1:1000,1:2000,1:5000,1:10000 and 1:50000) and added into the well, auristatin with 3 concentrations (10ng/ml,50ng/ml and 500ng/ml) is added into each gradient for competitive incubation while the antibodies are added, goat anti-mouse IgG for detecting HRP coupling is added for detection, and irrelevant antibodies are added for control detection. The results are shown in FIG. 1 and show that: 2F10, 4C3, 1G1 all affected their affinity for auristatin-coupled IgG after addition of auristatin and competed for an increase with increasing auristatin concentration, with 500ng of auristatin almost completely competing for antibody binding to the coupled auristatin at antibody 1:2000 dilution. Therefore, the anti-auristatin antibody has strong specificity and high sensitivity.

Example 3 detection of auristatin monoclonal antibodies auristatin (coupled auristatin and auristatin)

1) Establishment of competitive ELISA method for quantitative detection of auristatin molecules

In competitive ELISA, a high-specificity antibody is adsorbed on a solid carrier, so that free auristatin and a chemically synthesized auristatin conjugate (rabbit IgG coupled auristatin) compete for binding sites of the antibody in sequence, when the amount of the adsorbed antibody and the amount of the auristatin conjugate are fixed, the more free auristatin in a sample to be detected is, the less auristatin conjugate bound on the antibody is, the less free auristatin conjugate is bound with a goat anti-rabbit enzyme labeled secondary antibody, so that the color development of a substrate is lighter, and the amount of the auristatin to be detected and the absorbance value are in inverse proportion.

Through the matching of the coating antibody and auristatin coupled rabbit IgG and the optimized screening of the concentration of the detected secondary antibody, the operation flow of the competitive ELISA method is determined and is used for detecting the auristatin concentration in the serum of the rat. The procedure and results were as follows:

The operation procedure is as follows:

1) Coating monoclonal antibody: mixing purified monoclonal antibodies 2F10 and 4C3 with carbonate buffer solution (pH9.4-9.8) according to a molar ratio of 1:1, diluting to 5 mu g/ml, coating with 100 mu L/hole, and standing overnight (16-18 hours) at 2-8 ℃.

2) Washing the plate: the plate was washed with a washing solution (PBS + 0.05% Tween 20, pH 7.2-7.4) at 350. mu.L/well for 3 times.

3) and (3) sealing: blocking solution (1% BSA + 1% gelatin PBS solution) was added at 200. mu.L/well and blocked at 25. + -. 3 ℃ for 2 hours.

4) Washing the plate: reference step (2)

5) sample preparation: a standard curve: auristatin was diluted in rat serum to 0.25ng/mL, 0.5ng/mL, 1ng/mL, 2ng/mL, 4ng/mL, 8ng/mL, 16ng/mL, 32ng/mL auristatin standard solution as a test standard sample. B, standard curve: auristatin is diluted to be three concentrations of low, medium and high, namely 0.8ng/mL, 5ng/mL and 25ng/mL by rat serum to be used as a detection quality control sample.

6) Sample treatment: the standard curve sample, the quality control sample and the sample to be tested are pretreated with a diluent (1% BSA-PBS solution) at a ratio of 1:10 (e.g., 30. mu.L sample + 270. mu.L diluent → 300. mu.L).

7) Sample adding: all samples pretreated were applied to wells at 100. mu.L/well and incubated at 37 ℃ for 1 hour.

8) Adding the coupled auristatin: after washing the plate according to step (2), rabbit IgG conjugated auristatin 8. mu.g/mL, 100. mu.L/well was added and incubated at 37 ℃ for 1 hour.

9) Adding a detection secondary antibody: after washing the plate, adding HRP coupled goat anti-rabbit enzyme labeled antibody diluted 1:5000, 100 μ L/hole, and incubating at 37 ℃ for 1 hour.

10) Adding a substrate: and (3) after washing the plate according to the step (2), adding a newly prepared TMB substrate working solution, carrying out incubation at 100 mu L/hole for 10-15 minutes at room temperature in a dark place.

11) And (3) terminating the reaction: A2M solution of sulfuric acid was added at 50. mu.l/well.

12) And (3) detection: enzyme-linked immunosorbent assay (OD)₄₅₀And (6) reading.

13) And fitting the standard curve by adopting a four-parameter fitting method, calculating to obtain the concentration of each sample, and calculating the recovery rate of the standard curve and the quality control sample.

The results show that the range of the established quantitative standard curve of the rat serum auristatin is 0.25-32ng/mL, and the goodness of fit of each concentration point and the curve is good (the legend of the standard curve is shown in figure 2). Through multi-batch analysis, the reproducibility of the standard curve among batches is good, and the accuracy RE% range is as follows: RE is more than or equal to-2.4% and less than or equal to 5.2%; the repeatability among multiple holes of each concentration point is good, and the precision CV percent is less than or equal to 5.5 percent. The accuracy RE% range between the low, medium and high concentration quality control sample plates is as follows: RE is more than or equal to 13.5 percent and less than or equal to 13.1 percent, and the precision CV between plates is more than or equal to 10.2 percent. The data show that the method can be used for quantitative detection of auristatin in rat serum.

By adopting the same competition principle, the quantitative ranges of the established monkey and human serum auristatin standard curves are 0.39-50ng/mL and 0.625-40ng/mL respectively, and the method proves that the method can be applied to the quantitative detection of auristatin in the corresponding serum matrix.

2) Establishment of double-antibody sandwich ELISA method for coupling auristatin quantitative detection

The double-antibody sandwich ELISA is characterized in that a high-specificity antibody is adsorbed on a solid carrier, auristatin of an auristatin conjugate (human IgG coupled auristatin) is combined with a combination site of the antibody, the more the auristatin conjugate in a sample to be detected is, the more the auristatin conjugate combined on the antibody is, the more the auristatin conjugate is combined with a goat anti-human enzyme labeled secondary antibody, so that the deeper the color development of a substrate is, and the quantity of the auristatin conjugate to be detected is in direct proportion to the absorbance value.

Through the optimized screening of the concentration of the coating antibody and the concentration of the detection secondary antibody, the operation flow of the double-antibody sandwich ELISA method is determined and is used for detecting the concentration of the auristatin conjugate in the serum of the monkey. The procedure and results were as follows:

the operation procedure is as follows:

1) Coating monoclonal antibody: mixing purified monoclonal antibodies 2F10 and 4C3 with carbonate buffer solution (pH9.4-9.8) according to a molar ratio of 1:1, diluting to 4 mu g/mL, coating with 100 mu L/hole, and standing overnight (16-18 hours) at 2-8 ℃.

3) And (3) sealing: blocking solution (3% BSA-PBS solution) was added at 200. mu.L/well and blocked at 25. + -. 3 ℃ for 2 hours.

4) Washing the plate: reference step (2)

5) Sample preparation: a standard curve: auristatin conjugate was diluted in monkey serum in a gradient of 6.25ng/mL (anchor), 12.5ng/mL, 25ng/mL, 50ng/mL, 100ng/mL, 200ng/mL, 400ng/mL, 800ng/mL (anchor) of auristatin standard solution as a sample for detection. B, standard curve: and (3) diluting the auristatin conjugate into low, medium and high concentrations of 32ng/mL, 80ng/mL and 320ng/mL by using monkey serum to serve as a detection quality control sample.

6) Sample treatment: the standard curve sample, the quality control sample and the sample to be tested are pretreated with a diluent (1% BSA-PBS) at a ratio of 1:10 (e.g., 30. mu.L sample + 270. mu.L diluent → 300. mu.L).

8) Adding a detection secondary antibody: after washing the plate, adding HRP coupled goat anti-rabbit enzyme labeled antibody diluted 1:10000, 100 μ L/hole, and incubating for 1 hour at 37 ℃.

9) Adding a substrate: and (3) after washing the plate according to the step (2), adding a newly prepared TMB substrate working solution, carrying out incubation at 100 mu L/hole for 10-15 minutes at room temperature in a dark place.

10) And (3) terminating the reaction: A2M solution of sulfuric acid was added at 50. mu.L/well.

11) And (3) detection: enzyme-linked immunosorbent assay (OD)₄₅₀And (6) reading.

12) And fitting the standard curve by adopting a four-parameter fitting method, calculating to obtain the concentration of each sample, and calculating the recovery rate of the standard curve and the quality control sample.

The results show that the range of the established quantitative standard curve of the monkey serum auristatin is 12.5-400ng/mL, and the goodness of fit of each concentration point and the curve is good (the legend of the standard curve is shown in FIG. 3). Through multi-batch analysis, the reproducibility of the standard curve among batches is good, and the accuracy RE% range is as follows: RE is more than or equal to-0.9% and less than or equal to 3.8%; the repeatability among multiple holes of each concentration point is good, and the precision CV percent is less than or equal to 2.1 percent. The accuracy RE% range between the low, medium and high concentration quality control sample plates is as follows: RE is more than or equal to-10.8 percent and less than or equal to-4.3 percent, and the precision CV between plates is more than or equal to 10.5 percent. The data show that the method established by the auristatin antibody has high accuracy, and the method can be used for the quantitative detection of auristatin conjugates in monkey serum.

the method adopts a double-antibody sandwich ELISA method, the quantitative ranges of the established standard curves of the rat and human serum auristatin conjugates are respectively 6.25-800ng/mL and 39.06-500ng/mL, and the method verifies and shows that the method can be applied to the quantitative detection of auristatin conjugates in corresponding serum matrixes.

Claims

1. An auristatin antibody, or a functional fragment thereof, wherein the antibody comprises a heavy chain and a light chain, characterized in that:

(a) The heavy chain comprises heavy chain CDR1, CDR2 and CDR3 regions, wherein the heavy chain CDR1 sequence is an amino acid sequence shown in SEQ ID NO. 1, the heavy chain CDR2 sequence is an amino acid sequence shown in SEQ ID NO. 2, and the heavy chain CDR3 sequence is an amino acid sequence shown in SEQ ID NO. 3; the light chain comprises light chain CDR1, CDR2 and CDR3 regions, wherein the sequence of the light chain CDR1 is an amino acid sequence shown in SEQ ID NO. 4, the sequence of the light chain CDR2 is an amino acid sequence shown in SEQ ID NO. 5, and the sequence of the light chain CDR3 is an amino acid sequence shown in SEQ ID NO. 6; or

(b) The heavy chain comprises heavy chain CDR1, CDR2 and CDR3 regions, wherein the heavy chain CDR1 sequence is an amino acid sequence shown in SEQ ID NO. 7, the heavy chain CDR2 sequence is an amino acid sequence shown in SEQ ID NO.8, and the heavy chain CDR3 sequence is an amino acid sequence shown in SEQ ID NO. 9; the light chain comprises light chain CDR1, CDR2 and CDR3 regions, wherein the light chain CDR1 sequence is an amino acid sequence shown in SEQ ID NO. 10, the light chain CDR2 sequence is an amino acid sequence shown in SEQ ID NO. 11, and the light chain CDR3 sequence is an amino acid sequence shown in SEQ ID NO. 12; or

(c) The heavy chain comprises heavy chain CDR1, CDR2 and CDR3 regions, wherein the heavy chain CDR1 sequence is an amino acid sequence shown in SEQ ID NO. 13, the heavy chain CDR2 sequence is an amino acid sequence shown in SEQ ID NO. 14, and the heavy chain CDR3 sequence is an amino acid sequence shown in SEQ ID NO. 15; the light chain comprises light chain CDR1, CDR2 and CDR3 regions, wherein the sequence of the light chain CDR1 is the amino acid sequence shown in SEQ ID NO. 16, the sequence of the light chain CDR2 is the amino acid sequence shown in SEQ ID NO. 5, and the sequence of the light chain CDR3 is the amino acid sequence shown in SEQ ID NO. 17.

2. The auristatin antibody or functional fragment thereof according to claim 1, which comprises a heavy chain and a light chain, characterized in that:

(a) a heavy chain variable region which is the amino acid sequence shown in SEQ ID NO. 18; a light chain variable region which is the amino acid sequence shown in SEQ ID NO. 19; or

(b) A heavy chain variable region which is the amino acid sequence shown in SEQ ID NO. 20; a light chain variable region which is the amino acid sequence shown in SEQ ID NO. 21; or

(c) A heavy chain variable region which is the amino acid sequence shown in SEQ ID NO. 22; the variable region of the light chain which is the amino acid sequence shown in SEQ ID NO. 23.

3. An auristatin antibody or a functional fragment thereof according to any one of claims 1-2, wherein the antibody is a monoclonal antibody.

4. The auristatin antibody or functional fragment thereof according to any one of claims 1-2, wherein the functional fragment is Fv, scFv, Fab ', scFv-Fc, F (ab')2 fragment, Fv-PEG, scFv-PEG, Fab-PEG.

5. The auristatin antibody or functional fragment thereof according to claim 4, wherein the auristatin is MMAE.

6. A medicament, detection reagent or kit comprising an auristatin antibody or a functional fragment thereof according to any one of claims 1-2.

7. A method of detecting auristatin or a drug conjugate thereof for non-diagnostic or non-therapeutic purposes using the auristatin antibody or a functional fragment thereof described in any one of claims 1to 2.

8. The method of claim 7, wherein the auristatin antibody or the functional fragment is used in ELISA method.