CN107744592B

CN107744592B - anti-CD 56 antibody and dolastatin coupling compound and preparation method and application thereof

Info

Publication number: CN107744592B
Application number: CN201710840258.6A
Authority: CN
Inventors: 余琳; 姚于勤; 杨金亮
Original assignee: Sichuan University
Current assignee: Chengdu weisk biomedical Co.,Ltd.
Priority date: 2017-09-15
Filing date: 2017-09-15
Publication date: 2020-04-28
Anticipated expiration: 2037-09-15
Also published as: CN107744592A

Abstract

The invention belongs to the field of preparation of antibody coupling drugs, and particularly relates to a CD 56-resistant antibody and dolastatin coupling compound, and a preparation method and application thereof. The invention provides a conjugate compound of an anti-CD 56 antibody and dolastatin, which is characterized by comprising the following components: the anti-CD 56 antibody, the dolastatin, the coupling antibody and the connecting molecule of the dolastatin, one end of the coupling antibody and the connecting molecule of the dolastatin is coupled with the free sulfydryl of the anti-CD 56 antibody through maleimide, and the other end is coupled with the hydroxyl of the dolastatin through ester bond. The antibody coupling compound of the invention retains the high affinity ability of the anti-CD 56 antibody in targeting combination with CD56, has the ability of the dolastatin to kill cells efficiently, improves the treatment effect of the drug, reduces the toxic and side effects of the drug on the organism, can be used for preparing the drug for diagnosing and treating tumor, immune or nervous system diseases, and has good application prospect.

Description

anti-CD 56 antibody and dolastatin coupling compound and preparation method and application thereof

Technical Field

The invention belongs to the field of preparation of antibody coupling drugs, and particularly relates to a CD 56-resistant antibody and dolastatin coupling compound, and a preparation method and application thereof.

Background

Antibody-conjugated drugs (ADC) are novel conjugates which take Antibody targeting as a carrier and small-molecule cytotoxic warheads as effector molecules. The anti-tumor targeting peptide retains the specific targeting property of an antibody medicament and the efficient killing function of a small-molecule chemotherapeutic medicament, has the advantages of high anti-tumor activity and small toxic and side effects in vivo, and is a research hotspot of anti-tumor targeting medicaments.

The design of ADC drugs requires consideration of targets, cytotoxic small molecules and bioconjugation approaches. CD56, also known as Neural Cell addition Molecule 1(NCAM1), is one of the Neural Cell Adhesion molecules, is a type I membrane glycoprotein, and belongs to a member of the immunoglobulin superfamily. Its main biological functions are cell-to-cell adhesion and signaling. CD56 is bound to the effects on cell-cell adhesion through interactions between immunoglobulin domains of homophilics and cell-extracellular matrix interactions and cell signaling. CD56 is expressed in cells of the immune system, in nerve cells, in tumor cells, etc., but CD56 is highly expressed in a variety of primary and metastatic tumors, such as lung cancer, multiple myeloma, Merkel cell carcinoma, ovarian cancer, and brain glioma, etc. Wherein, the expression rate of CD56 in small cell lung cancer is 100%, the average surface of each small cell lung cancer cell has 15 ten thousand CD56 molecules, which is 60-100 times of normal tissue, closely related to cancer cell differentiation, invasion, metastasis and diffusion, and is an ideal target of ADC targeting chemotherapy drugs for cancers such as lung cancer. Meanwhile, because the CD56 is highly expressed on the surfaces of immune cells of some lesions and cells of neural origin, the CD56 may also be a target for treating some autoimmune and nervous system diseases.

Microtubule inhibitors exert their anti-tumor effects primarily by binding to tubulin. Dolastatin (MMA) is a polypeptide strong microtubule inhibitor, and there are two kinds of dolastatin, E and F (MMAE, MMAF), which are very high in cytotoxicity, IC50 is 10^-9-10-¹¹M, 100-fold stronger than doxorubicin. In the prior art, no report is available about whether the dolastatin is coupled with the anti-CD 56 antibody to be effective in resisting tumors.

Disclosure of Invention

The invention aims to solve the technical problem of providing a conjugate compound of an anti-CD 56 antibody and dolastatin, and a preparation method and application thereof.

The invention provides a conjugate compound of an anti-CD 56 antibody and dolastatin, which comprises the following components: anti-CD 56 antibody, aplysicin, a linker molecule coupling the antibody to aplysicin; one end of the linker molecule is coupled to the free thiol group of the anti-CD 56 antibody via maleimide, and the other end is coupled to the hydroxyl group of the haramicin via an ester bond.

In the anti-CD 56 antibody and dolastatin coupled complex, the free sulfydryl of the anti-CD 56 antibody is: free thiol groups are formed by opening disulfide bonds between or within antibody chains after reducing the light and heavy chains of the anti-CD 56 antibody with a reducing agent.

Furthermore, in the anti-CD 56 antibody and dolastatin conjugate complex, the reducing agent is at least one of dithiothreitol, β -mercaptoethanol or tris (2-carboxyethyl) phosphine.

Wherein, in the anti-CD 56 antibody and dolastatin coupling compound, the anti-CD 56 antibody comprises a heavy chain variable domain, a light chain variable domain and a constant region; the heavy chain variable domain is the same domain as the heavy chain complementarity determining region of the antibody, and the light chain variable domain is the same domain as the light chain complementarity determining region of the antibody.

In the anti-CD 56 antibody and dolastatin coupling compound, the amino acid sequence of the heavy chain variable domain of the anti-CD 56 antibody is any one of Seq ID NO 1, Seq ID NO 2, Seq ID NO 3, Seq ID NO 4, Seq ID NO 5 or Seq ID NO 6.

Furthermore, in the anti-CD 56 antibody-dolastatin conjugate complex, the amino acid sequence of the light chain variable domain of the anti-CD 56 antibody is any one of Seq ID NO. 7, Seq ID NO. 8, Seq ID NO. 9, or Seq ID NO. 10.

Furthermore, in the anti-CD 56 antibody and dolastatin conjugate complex, the constant region is a natural antibody constant region or a genetically engineered antibody constant region.

In the anti-CD 56 antibody and dolastatin coupled compound, the connecting molecule of the coupled antibody and dolastatin contains a valine structural unit and a citrulline dipeptide structural unit. The dipeptide in the connecting molecule can be degraded by cathepsin in cells to release the dolastatin to kill the cells.

Furthermore, in the anti-CD 56 antibody and dolastatin coupled complex, the structure of the connecting molecule of the coupled antibody and dolastatin is shown as formula 1.

Furthermore, in the anti-CD 56 antibody and dolastatin coupling compound, the dolastatin molecular structure is shown as formula 2 or formula 3

As shown.

Furthermore, in the anti-CD 56 antibody and dolastatin conjugate complex, the structure is formula 4 or formula 5.

In the anti-CD 56 antibody and dolastatin coupled compound, the molar ratio of the anti-CD 56 antibody to dolastatin is 1:1-10, and the molar ratio is preferably 1: 3.13.

The invention also provides a preparation method of the anti-CD 56 antibody and dolastatin coupled complex, which comprises the following steps:

a. preparing an anti-CD 56 antibody;

b. reducing the anti-CD 56 antibody by using a reducing agent to obtain an anti-CD 56 antibody with disulfide bonds on the surface;

c. adding dolastatin and coupling antibody and dolastatin linking molecule, reacting for 0.5-3h to obtain anti-CD 56 antibody and dolastatin coupling compound.

The third technical problem to be solved by the invention is to provide the application of the anti-CD 56 antibody and dolastatin coupled complex in preparing medicines for diagnosing or treating tumor, immune and nervous system diseases.

Wherein, Seq ID NO:1 anti-CD 56 antibody heavy chain variable domain 1; seq ID NO 2 anti-CD 56 antibody heavy chain variable domain 2; seq ID NO 3 anti-CD 56 antibody heavy chain variable domain 3; seq ID NO 4 anti-CD 56 antibody heavy chain variable domain 4; seq ID NO 5 anti-CD 56 antibody heavy chain variable domain 5; seq ID NO 6 anti-CD 56 antibody heavy chain variable domain 6; seq ID No 7 anti-CD 56 antibody light chain variable domain 1; seq ID NO 8 anti-CD 56 antibody light chain variable domain 2; seq ID NO 9 anti-CD 56 antibody light chain variable domain 3; seq ID No 10 anti-CD 56 antibody light chain variable domain 4; seq ID No. 11 nucleotide sequence encoding anti-CD 56 antibody heavy chain variable domain 1; seq ID No. 12 nucleotide sequence encoding anti-CD 56 antibody heavy chain variable domain 2; seq ID No. 13 nucleotide sequence encoding anti-CD 56 antibody heavy chain variable domain 3; seq ID No. 14 nucleotide sequence encoding anti-CD 56 antibody heavy chain variable domain 4; seq ID No. 15 nucleotide sequence encoding anti-CD 56 antibody heavy chain variable domain 6; seq ID No. 16 nucleotide sequence encoding anti-CD 56 antibody heavy chain variable structure 61; seq ID No. 17 nucleotide sequence encoding anti-CD 56 antibody light chain variable domain 1; seq ID No. 18 nucleotide sequence encoding anti-CD 56 antibody light chain variable domain 2; seq ID No. 19 nucleotide sequence encoding anti-CD 56 antibody light chain variable 31; seq ID No. 20 nucleotide sequence encoding anti-CD 56 antibody light chain variable domain 1.

The invention has the beneficial effects that: according to the invention, the anti-CD 56 antibody and the anti-CD 56 antibody coupling compound is obtained by cross-linking the dolastatin and the anti-CD 56 antibody through a biological coupling technology, and the antibody coupling compound can be combined with CD56 cells in a targeted manner, so that the utilization efficiency and the treatment effect of the medicine can be improved; the antibody coupling compound also has good affinity; the antibody coupling compound has high proliferation activity in vitro against CD56 positive tumor cells, has an anti-tumor effect, is an ADC candidate drug with strong targeting and high-efficiency tumor killing, can be used for preparing drugs for diagnosing and treating tumors, immune or nervous system diseases, and has good application prospects.

Drawings

FIG. 1 shows SDS-PAGE electrophoresis and Western-Blot detection of Promizimab purity, molecular weight, target recognition and antibody specificity

FIG. A, M is Marker, and lanes 1 to 6 are the extracellular end of human CD56 (CD56ECD), the cell line of small cell lung cancer NCI-H526, NCI-H524, NCI-H69, NCI-H128, and the expression of NCI-H446 in the non-reduced state in the cell lysate, and the corresponding sample in the reduced state in the cell lysate; b, M is Marker, 1-4 are respectively CD56 extracellular end non-reduction sample loading, CD56 extracellular end sugar removal non-reduction sample loading, CD56 extracellular end reduction sample loading, and CD56 extracellular end sugar removal reduction sample loading; C. the plot shows Promiximab cross-reactivity with CD56ECD as control, Promiximab cross-reactivity with human spleen cell lysate, NK cell lysate, and mouse spleen cell lysis (corresponding to lanes 1-4); and tested for tissue specificity in comparison to and commercial anti-CD 56 antibody 123C 3.

FIG. 2 shows the Western-Blot detection of the binding of the CD56 antibody to the lysis of CD56 positive cells

The Western-Blot detection results of CD56 antibodies 1-24 and CD56 positive cells NCI-H526 and NCI-H446(CD56 weak positive) show that the anti-CD 56 antibody can recognize other antibodies except that the CD56 linear epitope cannot be recognized by No. 2 antibody, wherein the first lane corresponding to each antibody is NCI-H526 lysate, and the second lane is NCI-H446 cell lysate.

FIG. 3A-X shows CD56 antibody affinity detection

The affinity of the 24 anti-CD 56 antibodies to the extracellular domain of human CD56 was analyzed using the Biolayer interferometry technique.

FIG. 4 shows the Promiximab affinity assay

Analysis by the Biolayer interference technique showed that the affinity of promiximab to CD56ECD was 0.78pmol (k)_a(1/Ms)＝1.27E+05,k_d(1/s)<1.0E-07,K_D<7.8E-13)。

FIG. 5 detection of PI value of Promiximab by capillary isopoint focusing method

The capillary isoelectric focusing method is used for detecting the PI value of the Promiximab, and the detected isoelectric point of the Promiximab which is expressed and purified by eukaryotic cells is 7.89.

FIG. 6 is a graph showing the detection of internalization by flow cytometry of the binding of the Promiximab targeting property of the anti-CD 56 antibody to CD56

FIG. A, Promiximab binding to CD56, cell membrane antigen of small cell lung carcinoma cell line, and incubation of small cell lung carcinoma with PBS (black part) and Promiximab (open line); FIG. B, Promizimab internalization efficiency assay, small cell lung carcinoma cell group with PBS (black area, 4 ℃,3h), Promiximab (solid line, 4 ℃,3h), Promiximab (dotted line, 37 ℃,3h), respectively; promizimab has better targeting in NCI-H526, NCI-H524 and NCI-H69 cell lines, and the internalization efficiency is 68.19%, 53.14% and 64.97% respectively. In conclusion, the prepared novel targeting CD56 antibody promiximab has specific binding capacity, higher affinity and effective internalization efficiency.

FIG. 7 is a diagram showing the examination of the in vitro activity of the anti-CD 56 antibody Promiximab with CCK-8

CCK-8 antiproliferative experimental results show that the IC50 of the Promiximab antibody on CD56 positive cell NCI-H69 is 968.54 +/-34.76 nM; the IC50 of the anti-CD 56 antibody against NCI-H526, a CD56 positive cell, was 792.24. + -. 41.21 nM.

FIG. 8 shows the in vivo anti-tumor activity of Promiximab.

A CD56 positive human small cell lung cancer NCI-H526 nude mouse subcutaneous tumor model is established, and the in vivo anti-tumor activity of promiximab is researched. The administration mode is tail vein, and the administration is once every two days and three times continuously. The results showed that the promiximab (10mg/kg) dose group showed significant in vivo activity of inhibiting tumor growth on day 24 in the NCI-H526 subcutaneous model. The observation of body weight shows that there is no significant difference in body weight between the control and the administered animals, which preliminarily indicates that toxicity of promiximab is small.

FIG. 9 shows toxicity analysis of Promiximab on heart, liver, spleen, lung and kidney

General status evaluation of mice was divided into body weight and pathological changes of major organs. On the 20 th day after tail vein administration, the heart, liver, spleen, lung and kidney of nude mice of each experimental group were taken and stained by H & E, and the results showed that the organs in the Chinese medicinal composition did not show obvious tissue lesion compared with the normal organs of the control group, indicating no obvious toxic or side effect.

FIG. 10 shows the drug-drug antibody coupling ratio for the detection of promiximab-MMAE by mass spectrometry, with an average antibody coupling ratio of 3.13;

FIG. 11 shows the detection of promiximab-MMAE by SDS-PAGE

In the figure, M is a protein Marker, 1 and 2 are promiximab without any treatment and promiximab subjected to denaturation reduction treatment respectively, 3 and 4 are promiximab subjected to TCEP treatment and promiximab subjected to denaturation reduction treatment respectively, 5 and 6 are promiximab-MMAE obtained by coupling reaction and promiximab-MMAE subjected to denaturation reduction treatment respectively;

FIG. 12 shows Proximab-MMAE binding capacity and targeting validation

The binding capacity of Promiximab after coupling with MMAE and the binding capacity of Promiximab-MMAE and small cell lung cancer cell lines are determined by flow cytometry, and the small cell lung cancer cells are incubated with PBS (shaded part), Promiximab (solid line) and Promiximab-MMAE (dotted line);

FIG. 13 shows the kinetic analysis of the affinity of Promizimab-MMAE

The binding constant of the promiximab-MMAE and CD56 is detected by measuring the affinity kinetics between different concentrations of promiximab-MMAE and the extracellular end of CD56 by using the biolayer interaction binding assay technology, and the promiximab-MMAE can be proved to be capable of effectively identifying and targeting CD 56.

FIG. 14 shows the in vitro cytotoxicity assays for Promiximab and Promiximab-MMAE

IC 72h after the small cell lung cancer cell line and the human NK cell and different concentrations of promiximab and promiximab-MMAE act in vitro by taking untreated cells as a control₅₀The value is obtained. The results show that Promiximab-MMAE also has strong cell killing effect on small cell lung cancer cell lines NCI-H526, NCI-H524 and NCI-H69, and the IC50 of the Promiximab-MMAE is 5.23nmol/L, 19.24nmol/L and 0.32nmol/L respectively.

FIG. 15 shows the evaluation of the efficacy of Promiximab and Promiximab-MMAE on subcutaneous transplantable tumors in vivo in mice with small cell lung cancer NCI-H526 and NCI-H69

Panel A and panel B show plots of tumor volume as a function of time following treatment of the subcutaneous tumor transplants of Promiximab and Promiximab-MMAE in NCI-H69 and NCI-H526 small cell lung carcinoma mice, respectively; panel C and panel D show graphs of nude mouse body weight over time after treatment of NCI-H69 and NCI-H526 small cell lung cancer mouse subcutaneous tumor transplantation with Promiximab and Promiximab-MMAE, respectively;

FIG. 16 is a pathological examination of the major organs of nude mice after treatment with Promiximab and Promiximab-MMAE.

Detailed Description

In the anti-CD 56 antibody and dolastatin coupled complex, the free sulfydryl of the anti-CD 56 antibody is: free thiol groups are formed by opening disulfide bonds between or within antibody chains after reducing the light and heavy chains of the anti-CD 56 antibody with a reducing agent. In order to release the aplysin, the connecting molecule selected by the invention contains a valine structural unit and a citrulline dipeptide structural unit, and the connecting molecule with the structural formula 1 is preferably selected.

The anti-CD 56 antibody of the present invention may be any antibody capable of targeting CD56, and preferably, an anti-CD 56 antibody in which the amino acid sequence of the variable domain of the heavy chain is any one of Seq ID No. 1, Seq ID No. 2, Seq ID No. 3, Seq ID No. 4, Seq ID No. 5 or Seq ID No. 6 and the amino acid sequence of the variable domain of the light chain is any one of Seq ID No. 7, Seq ID No. 8, Seq ID No. 9 or Seq ID No. 10, and the constant region of the antibody may be a natural antibody constant region or a genetically engineered antibody constant region, is used.

In particular, the anti-CD 56 antibody can be obtained by conventional hybridoma cell secretion, or can be constructed by conventional genetic recombination techniques. The resulting genes encoding the heavy and light chain variable domains of the CD56 antibody are cloned into Expression vectors with antibody constant region genes, including pAZ-V5-hCH, pAZ-V5-hCH, pAZ-V5-hCH, pAZ-V5-hCH, pAZ-V5-hCL, pAZ-V5-hCL, pTT5, pCEP4Mammalian Expression Vector, etc., expressed by eukaryotic Expression systems such as CHO and 293, etc., and CD56 antibody is obtained by protein purification methods.

Furthermore, aiming at the prepared anti-CD 56 antibody with strong targeting property, the anti-CD 56 antibody is coupled with the aplysiycin through the connecting molecules, can specifically target tumor cells, has strong affinity with the tumor cells, and can improve the utilization efficiency and the treatment effect of the aplysiycin.

The antibody conjugate of the present invention can be prepared into various forms of pharmaceutical preparations according to conventional pharmaceutical techniques, preferably injections, and most preferably lyophilized injections.

The anti-CD 56 antibody and dolastatin coupled compound can be used for preparing medicines for diagnosing or treating tumor, immune and nervous system diseases.

The following examples further illustrate specific embodiments of the present invention, but are not intended to limit the scope of the present invention to the examples.

The reagents and consumables used in the examples are all common commercial products.

EXAMPLE 1 preparation of CD56 antibody conjugate drug Proximab-MMAE

1. Preparation of anti-CD 56 antibody

And obtaining the light-heavy chain variable region amino acid sequence of the candidate clone through sequencing and sequence analysis, and selecting the sequence corresponding to each clone to construct a chimeric expression vector for the modification of the human-mouse chimeric antibody. The light chain variable region gene with the amino acid sequence shown as Seq ID NO:7 is constructed on a pTT5 expression vector, EcoR I-Kozak sequence-signal peptide-VH-constant region-stop codon-Hind III, and the light chain constant region gene sequence is as follows: NCBI Reference Sequence NG-000834.1, EcoRI-signal peptide-VL-Ig kappa chain C region-Hind III. Cloning the heavy chain variable region gene with the amino acid sequence shown as Seq ID NO:1 into an expression vector pTT 5/anti-CD 56 antibody-VH, wherein the expression vector consists of EcoR I-signal peptide-VH-Iggamma-1 chain C region-Hind III, and the heavy chain constant region sequence is as follows: NCBI Reference Sequence NG _ 001019.6. Carrying out codon optimization according to a mammalian cell expression system and synthesizing related gene segments. The antibody constructed by the above was named: the anti-CD 56 antibody Promiximab is prepared by a conventional method to obtain the anti-CD 56 antibody Promiximab.

2. Determination of anti-CD 56 antibody Promiximab Performance

(1) Detecting purity, molecular weight, target recognition and antibody specificity of Promiximab by SDS-PAGE electrophoresis and Western-Blot

Preparing a cell sample, collecting a small cell lung cancer cell line, resuspending with PBS, 3500rpm, centrifuging for 5min, washing for three times, adding a proper amount of cell lysate (containing protease inhibitor) according to the number of cells, fully and uniformly mixing, placing on ice for 30-60min, centrifuging for 10min at 4 ℃, 10000 ℃14000g, taking supernatant, subpackaging, labeling, storing at-20 ℃ for later use, wherein the pure protein sample addition concentration for SDS-PAGE is 3 mu g/hole, the sample addition system is 10 mu L, and the protein sample addition concentration is 0.1-100 ng/hole when the protein sample addition system is used for Wertern Blot detection. Mixing cell lysate (or protein sample) with 5 Xdenatured reducing sample buffer solution and 5 Xnon-denatured non-reducing sample buffer solution, mixing with denatured reducing sample buffer solution, standing at 100 deg.C for 5min, centrifuging at 10000rpm for 1min, and preparing electrophoresis sample. According to the size of the antigen molecular weight, the concentration of the separation gel is 8%, the concentration of the concentrated gel is 5%, gel is prepared, an electrophoresis system is installed, electrode buffer solution is added, sample loading is carried out, the voltage is stabilized to be 200V, the electrophoresis time is judged according to the position and the experiment purpose of bromophenol blue, after the electrophoresis is finished, a glass plate is detached, the gel is stripped, Coomassie brilliant blue is used for dyeing overnight, and after decoloration is carried out by adopting decoloration liquid, the SDS-PAGE electrophoresis result is analyzed. After performing the SDS-PAGE experiments according to the experimental procedure, one gel was analyzed by Coomassie blue staining and one gel was ready for use. Western-Blot membrane transfer according to the protocol, incubation antibody exposure.

The Promiximab is detected to have typical IgG structural domain characteristics, and the purity of the purified Promiximab is 96% through SDS-PAGE detection, and the Promiximab has typical light heavy chain structural domain (shown in figure 1A). The Western-Blot assay result shows (as shown in FIG. 1B) that Promiximab can be combined with pure CD56 and also can be combined with protein after CD56 positive cell lysis, and the antibody specificity experiment proves (as shown in FIG. 1C) that Promiximab has better specificity compared with the commercial antibody 123C 3.

The Western-Blot detection results of the antibodies and CD56 positive cells NCI-H526 and NCI-H446(CD56 weak positive) are shown in FIG. 2, the first lane corresponding to each antibody is NCI-H526 lysate, the second lane is NCI-H446 cell lysate, and the result shows that the anti-CD 56 antibody can recognize other antibodies except that the No. 2 antibody cannot recognize the CD56 linear epitope.

(2) BIACORE T200 avidity detection of avidity of anti-CD 56 antibodies

The affinity detection antigen is recombined Human NCAM-1/CD 56120 isoform with molecular weight of 120kD, and is coupled to CM5(BIACORE X100) or sCM5(BIACORE T200) through amino group by direct method; according to Rmax ═ MWanalyte/MWligand). times.RL × Sm, and the actual coupling amount is 1.5 times of that of RL. Calculating the coupling amount of the antigen suitable for detection at the same time to be 120 Ru; coupling operation: the coupling buffer was 1 XHBS-EP buffer, 50mM NaOH as wash. The antigen was formulated to 10. mu.g/mL using a sodium acetate solution of pH 4.5. Selecting amino coupling, inputting 120Ru into Target level, and placing an antigen sample, NaOH, ethanolamine, EDC, NSH and an empty test tube into corresponding positions in a sample tray according to prompts; the regeneration condition is groped, and glycine hydrochloric acid with the pH of 1.7 is finally selected for regeneration for 10 s; all samples are provided with multiple holes; setting a program, and selecting 2-1 by Flow pat in a Kinetics option; regeneration selection 2; contact time is 180 s; dissociation time was set to 5600 s; regeneration conditions are as follows: gly 1.7, 10 s; the samples tested in duplicate were spaced apart and three 0 concentrations and 3 start ups were set. The kinetic analysis was performed using a 1:1 computational model for fitting as shown in FIG. 3. The results showed that the affinity of Promiximab for CD56ECD was 0.78pmol (k)_a(1/Ms)＝1.27E+05,k_d(1/s)<1.0E-07,K_D<7.8E-13, as shown in FIG. 4), is a high affinity antibody. Other antibodies also showed stronger affinity.

(3) Detection of isoelectric point of Promiximab by capillary isoelectric focusing electrophoresis

Capillary electrophoresis isoelectric focusing is a method for performing analytical separation of samples according to the difference of pI values of the samples. The ampholyte is capable of forming a pH gradient within the capillary under the influence of a DC voltage across the two terminals. The samples with the amphiphatic groups migrate at different rates in the opposite direction to their charge. When the pH value of the sample is shifted to a pI value, the net charge of the sample is zero, and the movement is stopped. According to the principle, the aim of analyzing the pI value can be achieved.

To an EP tube was added 17.5. mu.L of 1% MC (final concentration of 0.35%), 2. mu.L of ampholyte pH 3-10 (final concentration of 4%), the corresponding volume of protein sample (final concentration of 0.2mg/ml) was added and 50. mu.L was made up with ultrapure water, vortexed with a vortex mixer for 30s, and centrifuged at 13,000rpm/min for 5min in a 4 ℃ centrifuge. The samples were added to a visual Insert Pack and centrifuged for 1min at 13,000rpm/min in a 4 ℃ centrifuge. Placing the visual Insert Pack into the visual Pack and then into a sample pan on the machine; operating according to the operating rules; focus Period 1 was set to 1500V,1min, FocusProperty 2 was set to 3000V,8 min. Carrier Amphibiytes: 4% pH 3-10, Additives: 0.35% MC, Concentration: 0.2 mg/ml. Promiximab expressed and purified by eukaryotic cells was determined to have an isoelectric point of 7.89 (shown in fig. 5).

(4) Detection of anti-CD 56 antibody Promiximab targeting by flow cytometry

Culturing CD56 positive tumor cell line, collecting cancer cells in logarithmic growth phase, dividing the cells into several groups with 1 × 10 cells in each group⁶(ii) a Grouping cells: negative control group (PBS group) and experimental group; resuspending each group of cells with PBS buffer salt, centrifuging at 4 deg.C and 3500rmp for 3min, and sucking the supernatant with the tip of a gun; incubating the primary antibody, adding 100 μ L PBS to the negative control group, adding 100 μ L antibody (10 μ g/mL) to the experimental group, resuspending, and incubating at 4 deg.C for 30 min; centrifuging each group of cells at 3500rpm for 3min, removing supernatant, adding 300 μ L PBS buffer salt into each group, resuspending and blowing the cells twice, centrifuging each group of cells at 3500rmp for 3min, removing supernatant, and repeating the steps for 2 times; incubating the secondary antibody, adding goat anti-human IgG/FITC labeled 50-100 μ L (dilution ratio of 1:200) into each group of cells, re-suspending and mixing, and incubating the cells at 4 ℃ for 30 min; after incubation at 4 ℃ for 30min is finished, adding 300 mu L PBS buffer salt into each group, resuspending and blowing the cells twice, centrifuging each group of cells at 3500rpm for 3min, sucking out supernatant, and repeating the steps for 2-3 times; adding 400 mu L PBS buffer solution to resuspend each group of cells for detection; on a flow cytometer, various parameters are set, cells of a control group are used for debugging and are used as negative control, and then cell samples of various groups are sequentially measured. The experimental results showed that Promiximab could better recognize and target the CD 56-positive small cell lung cancer cell lines NCI-H526, NCI-H524, and NCI-H69 (as shown in FIG. 6).

(5) Flow cytometry for detecting internalization effect of anti-CD 56 antibody Promiximab after binding to CD56

Culturing CD56 positive lung cancer cell line, collecting cells in logarithmic growth phase, dividing the cells into several groups with 1 × 10 cells in each group⁶Grouping cells: negative control group (PBS group) andthe experimental groups were resuspended in PBS buffer salt, centrifuged at 3500rmp for 3min at 4 ℃ and the supernatant aspirated by the tip of a gun. During internalization, the cells combined with the antibody are divided into two groups, one group is placed at 4 ℃ for 3h after the cells are resuspended by 500 mu LPBS, the other group is internalized at 37 ℃ for 3h, after internalization is finished, centrifugation is carried out at 4 ℃ and 3500RMP for 3min, the supernatant is sucked by a gun tip, 300 mu L of 50mM PBS buffer salt is added into each group, the cells are resuspended and blown twice, and then, the cells of each group are centrifuged at 3500RMP for 3min and the supernatant is sucked off. Repeating the previous step, adding goat anti-mouse IgG/FITC labeled secondary antibody (the dilution ratio is 1:200) to each group of cells, re-suspending and uniformly mixing, incubating the cells at 4 ℃ for 30min, injecting the primary antibody of promiximab, labeling the secondary antibody of goat anti-human IgG/FITC, adding 300 mu L of PBS buffer solution to each group, re-suspending and blowing the cells twice, centrifuging each group of cells at the rotation speed of 3500rpm for 3min, and sucking out the supernatant; repeating the steps for 2 times, adding 400 mu L PBS buffer solution to resuspend each group of cells for detection, setting each parameter on a flow cytometer, debugging the cells of a control group and taking the cells as negative control, and then sequentially measuring each group of cell samples. The experimental results showed that the silencing of internalization mediated by Promiximab binding to CD56 was 68.19%, 53.14% and 64.97% in NCI-H526, NCI-H524 and NCI-H69 cell lines, respectively. This result suggests that Promiximab has better CD56 targeting and at the same time has the ability to deliver drugs into CD56 positive cells (as shown in fig. 6).

(6) Activity assay of anti-CD 56 antibody Promiximab

CCK-8 was used to examine the in vitro activity of the anti-CD 56 antibody Promiximab

Culturing human small cell lung cancer cells NCI-H526 and NCI-H69 in RPMI 1640 culture medium containing 20% fetal calf serum, and culturing in 5% CO2 at 37 deg.C constant temperature cell culture box; collecting CD56 positive lung cancer cells in logarithmic growth phase, counting and adjusting cell density; laying 96-well plates, wherein the cell density of NCI-H526 and NCI-H69 is 10000/well and the volume is 100 mu L/well according to the growth speed of different cells; the cells were cultured in an incubator for 24 hours and then treated with chemicals. Diluting the antibody Promiximab with 20% fetal calf serum RPMI 1640 culture medium to initial concentration of 2.4 μ M, filtering with 0.22 μ M filter, and storing at 4 deg.C; add the gradient diluted sample to the 96-well plate plated with cells. 8 concentration gradients were designed for each drug, 3 duplicate wells were set for each concentration gradient, resulting in final antibody concentration gradients of 1.2. mu.M, 300nM, 75nM, 18.75nM, 4.69nM, 1.17nM, 0.29nM, 0.07nM, 0.02nM, and a dosing regime of 100. mu.L/well. The sample was added and gently tapped at the edge of the well plate and mixed, and the mixture was cultured in a 5% CO2 cell incubator at 37 ℃ for 72 hours. Observing the growth condition of the cells under a microscope every 24 hours; a96-well plate was filled with 20. mu.L/well of CCK-8 reagent (Dojindo) filtered through a 0.22 μm filter; incubating at 37 ℃ for 1-3h in the dark; the well plate was placed at 450nm in an enzyme linked immunosorbent assay, the OD of each well was measured and the average value for each group was calculated. The inhibition ratio (%) (control group-administered group)/control group × 100%. IC50 was calculated using GraphPad Prism 6.0. The experimental results showed (as shown in fig. 7) that the IC50 of the anti-CD 56 antibody against CD56 positive cells NCI-H69 was 968.54 ± 34.76 nM; the IC50 of the anti-CD 56 antibody against NCI-H526, a CD56 positive cell, was 792.24. + -. 41.21 nM.

In vivo antitumor Activity of Promiximab

The cells for cell culture and inoculation are cultured by using an RPMI 1640 culture medium containing 20% fetal calf serum to human CD56 positive lung cancer cell NCI-H526 and are placed in a 5% CO2 constant-temperature cell culture box for culture at 37 ℃; changing the liquid once 24 hours before inoculation; cells in the logarithmic growth phase were collected, washed twice with serum-free and antibiotic-free RPMI 1640 medium, and counted. Cell density was adjusted to 10 using serum-free, antibiotic-free RPMI 1640 medium and matrigel (final concentration > 4mg/mL)⁸Viable cells/ml.

The experimental animal is a BALB/c athymic nude mouse (nude mouse, Beijing Huafukang for short), the mouse is 5-6 weeks old, and the weight is 16-18 g; experimental animal feeding management: the breeding feed, drinking water, bedding and cages were changed once a week while observing the physiological state of the animals.

Cell inoculation: the inoculation amount is more than 1-2 multiplied by 10⁷Each cell, the inoculation volume is 100-; the tumor volume of the nude mice was observed every 3 days 7-15 days after the tumor cells were inoculated. When the tumor volume reaches 150mm3, grouping and administration can be carried out; picking up swellingNude mice with uniform tumor size were randomly grouped according to experimental purpose, and the groups of in vivo anti-tumor experiments in the NCI-H526 subcutaneous model are shown in table 1 below:

TABLE 1 NCI-H526 subcutaneous model grouping

NCI-H526 subcutaneous model grouping	Number (only)
		Control	6
Promiximab(10mg/kg)	6

Weighing the weight of each group of nude mice, measuring the tumor volume, and starting to administer the drug in a tail vein injection mode; the medicine is taken once every three days and is continuously taken for 3 times; monitoring the growth condition of the tumor of the nude mice: from the start of administration, the tumor length and length were measured every three days using a vernier caliper in mm, and the tumor volume was calculated as V ═ length × length/2. The experimental results show that the Promiximab has certain activity against CD56 positive tumors in vivo (as shown in FIG. 8).

Pathological examination of toxicity of Promiximab in vivo

Tissue samples of heart, liver, spleen, lung and kidney of control and antibody s drug-treated groups of nude mice were fixed in 4% phosphate-buffered formaldehyde and paraffin-embedded according to a conventional method. Tissue sections were stained with hematoxylin and eosin (H & E) and analyzed under the mirror to assess their destruction of normal tissues. The experimental results showed that no significant tissue lesions were observed in each organ of the Promiximab antibody group compared to the normal organs of the control group, indicating no significant toxic side effects (as shown in fig. 9).

3. Search for reaction conditions

(1) Establishment of standard curve of free sulfydryl of L-cysteine

Cysteine concentrations were prepared at 0.1mmol/L, 0.09mmol/L, 0.08mmol/L, 0.07mmol/L, 0.06mmol/L, 0.05mmol/L, 0.04mmol/L, 0.03mmol/L, 0.02 mmol/L; adding DTNB (2mg/mL, 5mmol/L) according to the molar ratio of L-cysteine to DTNB of 1:1.2 for color development; adding color development solution, incubating at 37 deg.C for 10-15min to obtain yellowish or yellow solution; measuring the 412nm absorption value by a spectrophotometer; and (4) inputting and formulating a standard curve according to the absorption values corresponding to the cysteine solutions with different concentrations.

(2) Reduction of interchain disulfide bonds in antibodies

To a solution of promiximab antibody (5mg/mL, 0.03125mmol/L) was slowly added TCEP (0.5mg/mL,1.74mmol/L) in a molar ratio of 1:3, and reacted at 37 ℃ at 120rpm for 3 hours.

Remarking: the reduction of the antibody is independent of the system and concentration, and is dependent only on the molar ratio of antibody to TCEP upon reaction.

And taking out the reaction solution, adding 10-20 times of water, centrifuging by adopting an Ultrafree 30K ultrafiltration centrifugal tube at 4000rpm to the volume of the reaction solution, and repeating twice to remove TCEP to obtain the CD56 antibody with the free disulfide bond. Determination of the number of free thiol groups of the antibody: taking 100 mu L of TCEP antibody removing solution, simultaneously taking the same amount of antibody, and replacing the buffer solution of the antibody into water; detecting the concentration by using Nano Drop 2000/2000 c; diluting the TCEP-treated antibody and the antibody not treated with TCEP to 1mg/L (0.00625mmol/L) according to the obtained concentration, wherein the system is 0.2mL, and when DTNB (2mg/mL, 5mmol/L) is used for color development, each antibody molecule generates 8 free sulfydryl groups according to reduction and the molar ratio of the antibody molecule to the DTNB is 1:1.2, then after the system V (0.00625mmol/L × 8 × 0.2mL × 1.2 ÷ 5) ═ 2.4 μ L of DTNB is added, incubating the mixture at 37 ℃ for 10-15min, and measuring the absorption value at 412 nm; and subtracting the absorption value of the antibody which is not subjected to reduction treatment from the absorption value of the reduced antibody, substituting the obtained absorption value into an L-cysteine free sulfhydryl standard curve, and finally calculating the number of free sulfhydryl generated by reduction.

(3) Antibody conjugation to MMAE

The antibody and MMAE are dissolved in DMF according to a certain molar ratio, mc-vc-MMAE is directly and slowly added into the antibody subjected to TCEP reduction treatment, after fully and uniformly mixing, the mixture is reacted at room temperature and 40rpm for a certain time, and the specific reaction molar ratio and the reaction time are groped according to the following table 2.

Table 2 table for setting reaction conditions

Taking out the reaction solution, and removing unreacted micromolecules remained in the reaction system by adopting a Desalting column HiTrapTM desaling 5 mL; adding the collected eluent into the rinsed ultrafiltration tube, centrifuging at the rotating speed of 4000rpm/min for 15-20min, pouring out the lower-layer liquid of the ultrafiltration tube, adding a new replacement buffer solution into the upper-layer tube, fully and uniformly mixing the protein sample in the inner tube, repeating the steps for 1-2 times, and replacing the buffer solution of the ADCs sample with an ADCs sample preservation solution; the samples were sent to Waters for DAR analysis by mass spectrometry to obtain the optimum charge ratio and coupling reaction time.

By groping, the following results can be obtained: after reducing the inter-chain disulfide bonds of the antibody according to the above conditions, the effect is better when the molar ratio of the promiximab to the small molecule drug MMAE is 1:3.13 for coupling reaction for more than 3 hours.

4. Preparation of anti-CD 56 antibody and dolastatin coupling compound Promiximab-MMAE

Antibody promiximab (5mg/mL, 0.03125mmol/L) and TCEP (0.5mg/mL,1.74mmol/L) were mixed in a molar ratio of 1:3, slowly adding TCEP, reacting for 3h at 37 ℃ and 120rpm (cell shaker); after the reduction reaction is finished, taking out 100uL of reaction solution, calculating and reducing to generate free sulfydryl in the same step as before when the TCEP in the reaction solution is removed, and using the rest antibody reaction solution for subsequent coupling reaction (without removing the TCEP in the reaction solution); the molar ratio of the antibody to the small molecule drug MMAE is 1:3.13, directly and slowly adding mc-vc-MMAE dissolved in DMF into the rest TCEP-treated antibody reaction solution (white precipitate is generated after adding), fully and uniformly mixing, and reacting for 3h at room temperature and 40 rpm; after the coupling is finished, taking out reaction liquid, and removing residual unreacted micromolecules in a reaction system by adopting 5mL of a desalting column HiTratpTMDesaling; adding the collected eluent into an ultrafilter tube after rinsing, centrifuging at the rotating speed of 4000rpm/min for 5-10min, pouring out the lower-layer liquid of the ultrafilter tube, adding a new replacement buffer solution into the upper-layer tube, fully and uniformly mixing the protein sample in the inner tube, repeating the steps for 1-2 times, replacing the buffer solution of the ADCs sample with an ADCs sample preservation solution, and sending the rest sample to a Shanghai detection center of Waters company for analyzing the DAR of the sample by mass spectrometry. The average DAR of the prepared anti-CD 56 antibody and dolastatin conjugate complex Promiximab-MMAE was 3.13 as shown in fig. 10.

Example 2 anti-CD 56 antibody-conjugated drug Proximab-MMAE binding, targeting, and affinity assays

1. SDS-PAGE verifies the structure and purity of Promiximab-MMAE

The TCEP reduction-treated antibody promiximab and the small-molecule chemotherapeutic drug are coupled for 3 hours at room temperature (40rpm) according to the molar ratio of 1:6 to obtain the promiximab-MMAE ADCs compound. In order to prove that the CD56 antibody can still retain the original protein structure after the bioconjugation process, SDS-PAGE is used for verification, and since the small-molecule DUBA has small contribution to the whole molecular mass of the whole antibody Promiximab-MMAE conjugate, if the Promiximab antibody still keeps similar electrophoretic behavior with the unconjugated antibody after the bioconjugation operation, the protein structure of the Promiximab antibody is not greatly influenced by the bioconjugation process.

The experimental procedure was as follows: taking each analyzed protein sample, mixing with 5 times denatured reduced sample buffer solution and 5 times non-denatured non-reduced sample buffer solution uniformly, mixing with the denatured reduced sample buffer solution, placing at 100 ℃ for 5min, centrifuging at 10000rpm for 1min, and preparing an electrophoresis sample. The pure protein loading concentration for SDS-PAGE is 3 mug/hole, and the loading system is 10 mug. According to the size of the antigen molecular weight, the concentration of the separation gel is 8-12%, the concentration of the concentrated gel is 5%, gel is prepared, an electrophoresis system is installed, electrode buffer solution is added, the sample is loaded, the voltage is stabilized to be 200V, the electrophoresis time is judged according to the position and the experiment purpose of bromophenol blue, after the electrophoresis is finished, a glass plate is detached, the gel is stripped, Coomassie brilliant blue is used for dyeing overnight, and after decoloration is carried out by adopting decolorant, the SDS-PAGE electrophoresis result is analyzed.

The results are shown in fig. 11, and the molecular weight of Promiximab before and after the bioconjugation process is not significantly changed, and the protein structural fragments unique to the antibody are still maintained. The results show that the Promiximab-MMAE prepared by the biological coupling technology has similar antibody fragments with the Promiximab antibody, such as protein fragmentation and degradation.

2. Detection of targeting property of CD56 antibody-coupled drug Promiximab-MMAE by flow cytometry

Culturing CD56 positive small cell lung cancer cell line, collecting small cell lung cancer cell line in logarithmic phase, dividing the cells into several groups with 1 × 10 cells in each group⁶(ii) a Grouping cells: a negative control group (PBS group), a positive control group (huN901), and an experimental group; resuspending each group of cells with PBS buffer salt, centrifuging at 4 deg.C and 3500rmp for 3min, and sucking the supernatant with the tip of a gun; incubating the primary antibody, adding 100 μ L PBS to the negative control group, adding 100 μ L antibody huN901 (with the concentration of 10 μ g/mL) to the experimental group, resuspending, and incubating at 4 deg.C for 30 min; centrifuging each group of cells at 3500rpm for 3min, removing supernatant, adding 300 μ L PBS buffer salt into each group, resuspending and blowing the cells twice, centrifuging each group of cells at 3500rmp for 3min, removing supernatant, and repeating the steps for 2 times; incubating the secondary antibody, adding goat anti-human IgG/FITC labeled 50-100 μ L (dilution ratio of 1:200) into each group of cells, re-suspending and mixing, and incubating the cells at 4 ℃ for 30 min; after incubation at 4 ℃ for 30min is finished, adding 300 mu L PBS buffer salt into each group, resuspending and blowing the cells twice, centrifuging each group of cells at 3500rpm for 3min, sucking out supernatant, and repeating the steps for 2-3 times; adding 400 mu L PBS buffer solution to resuspend each group of cells for detection; on a flow cytometer, various parameters are set, cells of a control group are used for debugging and are used as negative control, and then cell samples of various groups are sequentially measured.

The experimental results are shown in fig. 12: after coupling the small-molecule drug MMAE, the binding capacity of the antibody promiximab to CD56 and the binding capacity of the antibody to CD56 positive cells are not changed, the binding capacity of the promiximab-MMAE and the promiximab to small-cell lung cancer cell lines is the same, and the binding capacity of the antibody is not influenced by the coupling of the small-molecule drug.

3. BIACORE T200 detection of CD56 antibody-conjugated drug Promiximab-MMAE affinity

The antigen is Recombinant Human NCAM-1/CD 56120 isofomm, the molecular weight is 120kD, and the antigen is coupled to CM5(BIACORE X100) or sCM5(BIACORE T200) through an amino group by adopting a direct method; according to the Rmax (MWanalyte/MWligand) multiplied by RL multiplied by Sm, the actual coupling amount is 1.5 times of RL. Calculating the coupling amount of the antigen suitable for detection at the same time to be 120 Ru; coupling operation: the coupling buffer was 1 XHBS-EP buffer, 50mM NaOH as wash. The antigen was formulated to 10. mu.g/mL using a sodium acetate solution of pH 4.5. Selecting amino coupling, inputting 120Ru into Target level, and placing an antigen sample, NaOH, ethanolamine, EDC, NSH and an empty test tube into corresponding positions in a sample tray according to prompts; the regeneration condition is groped, and glycine hydrochloric acid with the pH of 1.7 is finally selected for regeneration for 10 s; antibody huN901 preparation by diluting the antibody to 3.2nM, 1.6nM, 0.8nM, 0.4nM, 0.2nM, 0.1nM, 0.05nM and 0.025nM, 13,000rpm/min with 1 XHBS-EP buffer and centrifuging for 3 min; all samples are provided with multiple holes; hybridoma supernatant sample preparation: collecting hybridoma cell supernatant, centrifuging at 13,000rpm/min for 3min, detecting stock solution, and arranging multiple holes in each sample; setting a program, and selecting 2-1 by Flow pat in a Kinetics option; regeneration selection 2; contact time is 180 s; dissociation time was set to 5600 s; regeneration conditions are as follows: gly 1.7, 10 s; the samples tested repeatedly were spaced apart and three 0 concentrations and 3 start ups were set and the program was run with the click started. And selecting a proper concentration corresponding to a response unit change curve according to the test result, fitting by using a 1:1 calculation model, and performing kinetic analysis.

The results of the Biolayer interaction affinity assay are shown in FIG. 13 and show: the promiximab-MMAE prepared by this example has similar kinetic affinity characteristics to promiximab with the antigen CD 56. These results indicate that the conjugated small molecule drug has no effect on the binding ability of the antibody promiximab, and that promiximab-MMAE still has the same binding and recognition ability with CD56 as promiximab.

Example 3 CD56 antibody conjugate drugs anti-tumor Activity

1. Proximab-MMAE in vitro cytotoxicity detection

Culturing human small cell lung cancer cells NCI-H446, NCI-H526, NCI-H524, NCI-H69 and NCI-H128 in RPMI 1640 culture medium containing 20% fetal calf serum, and culturing in 5% CO2 at 37 deg.C constant temperature cell culture box; separating and extracting NK cells; collecting small cell lung cancer cells in logarithmic growth phase, counting and adjusting cell density; laying a 96-pore plate, wherein the density of NCI-H128 cells is 1000-1500 cells/pore, the density of NCI-H526, NCI-H524, NCI-H69 and NK cells is 10000 cells/pore and the volume is 100 mu L/pore according to the growth speed of different cells; the cells were cultured in an incubator for 24 hours and then treated with chemicals.

Respectively diluting the antibody and the ADCs compound with a culture medium containing 20% fetal bovine serum RPMI 1640 to an initial concentration of 2.4 μ M, filtering with a 0.22 μ M filter, and storing at 4 ℃; add the gradient diluted sample to the 96-well plate plated with cells. Since each well contains 100. mu.l of the medium, the initial concentration of the sample to be tested is 1.2. mu.M. 8 concentration gradients were designed for each drug, 3 duplicate wells were set for each concentration gradient, resulting in final concentration gradients of antibody and ADCs complexes of 1.2. mu.M, 300nM, 75nM, 18.75nM, 4.69nM, 1.17nM, 0.29nM, 0.07nM, 0.02nM, and a dosing regime of 100. mu.L/well. The sample was added and gently tapped at the edge of the well plate and mixed, and the mixture was cultured in a 5% CO2 cell incubator at 37 ℃ for 72 hours. Observing the growth condition of the cells under a microscope every 24 hours; to the 96-well plate of the above step, 20. mu.L/well of CCK-8 reagent (Dojindo Co.) filtered through a 0.22 μm filter; incubating at 37 ℃ for 1-3h in the dark; the well plate was placed at 450nm in an enzyme linked immunosorbent assay, the OD of each well was measured and the average value for each group was calculated. The inhibition ratio (%) (control group-administered group)/control group × 100%. IC50 was calculated using GraphPad Prism 6.0 [107 ].

The in vitro anti-proliferation experimental study of the Promiximab-MMAE and the Promiximab on the small cell lung cancer cell line and the human NK cell is to observe the cell killing effect by naked eyes and detect by adopting CCK-8 after the small cell lung cancer cell line and the human NK cell are acted for 72 hours in vitro.

The results showed that the in vitro killing IC50 values of promiximab-MMAE versus NCI-H526, NCI-H524, and NCI-H69 were 5.23. + -. 1.4nmol/L, 19.24. + -. 1.38nmol/L, and 0.32. + -. 0.1nmol/L, respectively (as shown in FIG. 14 and Table 3).

TABLE 3 Promiximab-MMAE in vitro cytotoxicity assay results

Although CD56 is also a marker molecule of NK, and promiximab can be combined with CD56 expressed on the surface of NK cells, the level of CD56 molecules on the surface of tumor cells is 60 times higher than that of NK cells, so the in vitro killing effect of promiximab-MMAE on NK cells is not obvious, and the IC50 is more than 1200 nmol/L. These results indicate that promiximab-MMAE has strong in vitro killing activity against CD56 positive small cell lung cancer cell line.

2. In vivo antitumor Activity of Promiximab-MMAE

Culturing human small cell lung cancer cells NCI-H526 and NCI-H69 in RPMI 1640 culture medium containing 20% fetal calf serum, and culturing in 5% CO2 at 37 deg.C constant temperature cell culture box; changing the liquid once 24 hours before inoculation; cells in the logarithmic growth phase were collected, washed twice with serum-free and antibiotic-free RPMI 1640 medium, and counted. The cell density was adjusted to 108 viable cells/mL using serum-free and antibiotic-free RPMI 1640 medium and matrigel (final concentration greater than 4mg/mL), the cell density calculation may not be accurate due to the growth of small cell lung cancer cell lines NCI-H526, NCI-H69 clumps, the seeding density was 1>10⁷。

The experimental animal is a BALB/c athymic nude mouse (nude mouse, Beijing Huafukang for short), the mouse is 5-6 weeks old, and the weight is 16-18 g; the experimental animals are generally 4-5 weeks old when they arrive at the goods, and need to be adaptively raised in an animal room for 1 week; experimental animal feeding management: the breeding feed, drinking water, bedding and cages were changed once a week while observing the physiological state of the animals.

Cell inoculation: the inoculation amount is more than 1-2 multiplied by 10⁷Each cell, the inoculation volume is 100-; the tumor volume of the nude mice was observed every 3 days 7-15 days after the tumor cells were inoculated. When the tumor volume reaches 150mm³Grouping and administration can be performed; nude mice with uniform tumor size were picked and randomly grouped according to experimental purpose inIn the NCI-H526 subcutaneous model, the experimental grouping of promiximab-MMAE in vivo anti-tumor experiments is shown in the following Table 4:

TABLE 4 NCI-H526 subcutaneous model grouping

NCI-H526 subcutaneous model grouping	Number (only)
		Control	6
Promiximab(10mg/kg)	6
		Promiximab-MMAE(10mg/kg)	6
Promiximab-MMAE(5mg/kg)	6
		Promiximab-MMAE(2.5mg/kg)	6
Promiximab-MMAE(1mg/kg)	6

In the NCI-H69 subcutaneous model, the groupings of the promiximab-MMAE ADCs in vivo antitumor experiments are shown in Table 5 below:

TABLE 5 NCI-H69 subcutaneous model group

NCI-H69 subcutaneous model grouping	Number (only)
		Control	6
Promiximab(10mg/kg)	6
		Promiximab-MMAE(10mg/kg)	6
Promiximab-MMAE(5mg/kg)	6
		Promiximab-MMAE(2.5mg/kg)	6

Weighing the weight of each group of nude mice, measuring the tumor volume, and starting to administer the drug in a tail vein injection mode; the medicine is taken once every three days and is continuously taken for 3 times; monitoring the growth condition of the tumor of the nude mice: measuring the length and the short diameter of the tumor by using a vernier caliper every three days from the beginning of administration, wherein the unit is mm, calculating the volume of the tumor, wherein the calculation formula is V (the length is multiplied by the short diameter) is 2, wiping the outline of the tumor by using an alcohol cotton ball when measuring the length and the short diameter, taking the rule as good as the tumor can pass through and the rule cannot be clamped, averaging the measured data and calculating the standard error; monitoring the weight of the nude mice, weighing the weight of the animals in g by using an electronic balance, averaging the measured data of each group, and calculating a standard deviation; observing the physiological and living states of the nude mice, and the treatment period such as the existence of infection: the treatment period depends on the treatment effect and the growth condition of the nude mice. Generally has obvious treatment effect, and the tumor volume of the control mice is more than 2000mm³In case, treatment was terminated and the mice were sacrificed.

CD 56-positive human small cell lung cancer NCI-H526 and NCI-H69 two nude mouse subcutaneous tumor models were used to study the in vivo anti-tumor activity of promiximab and promiximab-MMAE.

The results are shown in FIGS. 15A and B: in the NCI-H526 subcutaneous model, the promiximab-MMAE (10mg/kg) dose group completely regressed the tumor without recurrence after three administrations. In the NCI-H69 subcutaneous model, the promiximab-MMAE (10mg/kg) dose group can completely regress the tumor without recurrence after three times of administration, the tumor of three nude mice in the promiximab-MMAE (5mg/kg) dose group is completely regressed, and the observation result of the body weight shows (as shown in figures 15C and D) that the body weight of the nude mice is not obviously changed after administration, so the promiximab-MMAE compound prepared in the research has good in-vivo anti-CD 56 positive tumor activity and low toxic and side effect.

3. H & E staining

General status evaluation of mice was divided into body weight and pathological changes of major organs. H & E staining of major organs of tumor nude mice was used for potential toxic side effects study of promiximab-MMAE. Tissue samples of heart, liver, spleen, lung and kidney of control, antibody and ADCs drug-treated groups of nude mice were fixed in 4% phosphate-buffered formaldehyde and paraffin-embedded according to a conventional method. Tissue sections were stained with hematoxylin and eosin (H & E) and analyzed under the mirror. On the 20 th day after tail vein administration, the heart, liver, spleen, lung and kidney of nude mice of each experimental group were taken and stained with H & E, and the results are shown in fig. 16, and compared with normal organs of the control group, no obvious tissue lesion was found in each organ of the administered group, indicating no obvious toxic or side effect.

The embodiment shows that the anti-CD 56 antibody compound prepared by the invention not only retains the high affinity ability of targeting combination with CD56, but also has the ability of high-efficiency killing cells by aplysicin, and reduces the toxic and side effects of the drug on the organism while improving the therapeutic effect of the drug; the proliferation activity of anti-CD 56 positive tumor cells in vitro is high, the anti-tumor effect is achieved, the toxic and side effects are small, and a brand new choice is provided for ADCs.

Sequence listing

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<213> Artificial Sequence (Artificial Sequence)

<400>7

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

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Leu His Ser

20 25 30

Asn Gly Asn Thr Tyr Phe Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210>8

<211>107

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>8

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

1 5 10 15

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

20 25 30

Leu His Trp Tyr Gln Gln Pro Ser Asn Glu Ser Pro Arg Leu Leu Ile

35 40 45

Arg Tyr Val Cys Leu Ala Ile Ser Gly Ile Pro Ser Ser Phe Ala Asp

50 55 60

Ser Gly Ser Arg Thr Asp Phe Pro Leu Cys Ile Asn Ser Val Lys Pro

65 70 75 80

Glu His Val Gly Val Tyr Tyr Cys Gln Asn Gly His Thr Phe Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Val Lys

100 105

<210>9

<211>107

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>9

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

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Glu Asn Ile Gly Thr Ser

20 25 30

Met His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser

65 70 75 80

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Thr Asn Ser Trp Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Asn Leu Glu Ile Lys

100 105

<210>10

<211>107

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>10

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

1 5 10 15

Asp Ser Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Arg Asn Asn

20 25 30

Leu His Trp Tyr Gln Gln Gln Ser His Glu Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Thr

65 70 75 80

Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Asn Asn Trp Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210>11

<211>346

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

gagatccagc tgcagcagtc tggacctgag ctggtgaagc ctggggcttc agtgaaggta 60

tcctgcaagg cttctggtta tgcgttcact aactacaaca tgtactggat gaaacagagc 120

catggaaaga gccttgagtg gattggatat attgatcctt acaatggtgg tactaggtac 180

aaccagaagt tcaagggcaa ggccacattg actgttgaca agtcctccag cacagcctac 240

atgcatctca acagcctgac atctgaggac tctgcagtct attactgtgc aagagaggat 300

agtaccggct actggggcca aggcaccact ctcacagtct cctcag 346

<210>12

<211>360

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>12

cagatccagt tggtgcagtc tggacctgaa ctgaagaagc ctggagagac agtcaagatc 60

tcctgtaagg cttctggata taccttcaca aactatggaa tgaactgggt gaagcagact 120

ccaggaaagg gtttaaagtg gatgggctgg ataaacacct atactggaga gtcaacatat 180

actgatgact tcaagggacg gtttgccttt tctttggaga cctctaccag caccgcctat 240

ttgcagatca acaacatcag aaatgaggac acggctacat atttctgtgc aagatcccct 300

tattactacg gtagtcaacg ggggtacttc gatgtctggg gcgcagggac cacggtcacc 360

<210>13

<211>346

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>13

aaacgggtgg agtctggggg aggcttagtg cagcctggag ggtcccggaa actctcctgt 60

gcagcctctg gattcacttt cagtgactac ggaatggcgt gggttcgaca ggttccaggg 120

aaggggcctg agtggattgc attcattagt aatttggcat atagtatcta ctatatagac 180

actgtgacgg gccgattcac catctctaga gagaatgcca agaacaccct gtacctggaa 240

atgagcagtc tgaggtctga ggacacagcc atatactact gtgcaagggt ttctgggacc 300

tggcttggtt actggggcca agggactctg gtcactgtct ctgcag 346

<210>14

<211>339

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>14

tggagtctgg gggaggctta gtgcagcctg gagggtcccg gaaactctcc tgtgcagcct 60

ctggattcac tttcagtgac tccggaatgg cgtgggttcg acaggctcca gggaaggggc 120

ctgagtgggt agcattcatt agtaatttgg catatagtat ctactatgca gacactgtga 180

cgggccgatt caccatctct agagagaatg ccaagaacac cctgtacctg gaaatgagca 240

gtctgaggtc tgaggacaca gccatgtact actgtgcaag gatctcctat gattacattg 300

actactgggg ccaaggcacc actctcacag tctcctcag 339

<210>15

<211>351

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>15

gacgtgatgc tggtggagtc tgggggaggc ttagtgcagc ctggagggtc ccggaaactc 60

tcctgtgcag cctctggatt cactttcagt gactacggaa tggcgtgggt tcgacaggtt 120

ccagggaagg ggcctgagtg gattgcattc attagtaatt tggcatatag tatctactat 180

atagacactg tgacgggccg attcaccatc tctagagaga atgccaagaa caccctgtac 240

ctggaaatga gcagtctgag gtctgaggac acagccatat actactgtgc aagggtttct 300

gggacctggc ttggttactg gggccaaggg actctggtca ctgtctctgc g 351

<210>16

<211>344

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>16

ggtccagctg caacagtctg gacctgaact ggtgaagcct ggggcttcag tgaagatatc 60

ctgcaaggct tctggttact cattcactga ctactacatg cactgggtga agcaaagcca 120

tgtaaagagc cttgagtgga ttggacgtat taatccttac aatggtgcta ctacctacaa 180

ccagaatttc aaggacaagg ccagtttgac tgtagataag tcctccagca cagtctacat 240

ggagctccac agcctgacat ctgaggactc tgcagtctat tactgtgcaa gatacccttt 300

gtttggttac tggggccaag ggactctggt cactgtctct gcag 344

<210>17

<211>337

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>17

gatgttttgc tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60

atctcttgca gatctagtca gaatatttta catagtaatg gcaacaccta tttcgaatgg 120

tacctgcaga aaccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180

tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240

agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acatgttccg 300

ttcacgttcg gaggggggac caagctggaa ataaaac 337

<210>18

<211>322

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>18

gacattatta tatctcgttc tccagccacc ctgtctgtga ctccaggaga tagagtctct 60

ctttcctgca gggccagtct gattattacc gactacttac actggtatca acaaccatca 120

aatgaatctc caaggcttct catcagatat gtttgcctgg ccatctctgg gatcccctcc 180

tccttcgctg acagtggatc acggacagat ttccctctct gtatcaacag tgtgaaacct 240

gaacatgttg gagtgtatta ctgtcaaaat ggtcacacct ttccgctcac gttcggtgct 300

gggaccaagc tggaagtgaa ac 322

<210>19

<211>322

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>19

gacatcttgc tgactcagtc tccagccatc ctgtctgtga gtccaggaga aagagtcagt 60

ttctcctgca gggccagtga gaacattggc acaagcatgc actggtatca gcaaagaaca 120

aatggttctc caaggcttct cataaagtat gcttctgagt ctatctctgg gatcccttcc 180

aggtttagtg gcagtggatc agggacagat tttactctta gcatcaacag tgtggagtct 240

gaagatattg cagattatta ctgtcaacaa actaatagct ggccgtacac gttcggaggg 300

gggaccaacc tggaaataaa ac 322

<210>20

<211>322

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>20

gacgttgtgg taactcagtt tccagacacc ctgtctgtga ctccaggaga tagcgtcagt 60

ctttcctgca gggccagcca aagtattcgt aacaacctac actggtatca acaacaatca 120

catgagtctc caaggcttct catcaagtat gcttcccagt ccatctctgg gatcccctcc 180

aggttcagtg gcagtggatc agggacagat ttcactctca gtatcaacag tgtggagact 240

gaagattttg gaatgtattt ctgtcaacag agtaacaact ggccgtacac gttcggaggg 300

gggaccaagc tggaaataaa ac 322

Claims

1. The anti-CD 56 antibody and dolastatin coupling complex is characterized by comprising the following components: anti-CD 56 antibody, dolastatin, a linker molecule coupling the antibody to dolastatin; one end of the connecting molecule is coupled with free sulfydryl of the anti-CD 56 antibody through maleimide, and the other end is coupled with hydroxyl of the dolastatin through ester bond;

the anti-CD 56 antibody comprises a heavy chain variable domain, a light chain variable domain, a constant region; the heavy chain variable domain is the same domain as the heavy chain complementarity determining region of the antibody, and the light chain variable domain is the same domain as the light chain complementarity determining region of the antibody;

the amino acid sequence of the heavy chain variable domain of the anti-CD 56 antibody is Seq ID NO 1;

the amino acid sequence of the light chain variable domain of the anti-CD 56 antibody is Seq ID NO 7;

the connecting molecular structure of the coupling antibody and the dolastatin is shown as a formula 1

As shown.

2. The anti-CD 56 antibody-dolastatin conjugate complex of claim 1, wherein: the free thiol group of the anti-CD 56 antibody is: free thiol groups are formed by opening disulfide bonds between or within antibody chains after reducing the light and heavy chains of the anti-CD 56 antibody with a reducing agent.

3. The anti-CD 56 antibody-dolastatin conjugate complex of claim 2, wherein the reducing agent is dithiothreitol, β -mercaptoethanol, or tris (2-carboxyethyl) phosphine.

4. The anti-CD 56 antibody-dolastatin conjugate complex of claim 1, wherein: the constant region is a natural antibody constant region or an antibody constant region after genetic engineering.

5. The anti-CD 56 antibody-dolastatin conjugate complex of claim 1, wherein: the structure of the dolastatin molecule is shown as formula 2

Or formula 3

As shown.

6. The anti-CD 56 antibody-dolastatin conjugate complex according to any one of claims 1 to 5, wherein: the structure is formula 4

Or formula 5

7. The anti-CD 56 antibody-dolastatin conjugate complex of claim 1, wherein: the molar ratio of the anti-CD 56 antibody to the dolastatin is 1: 1-10.

8. The anti-CD 56 antibody-dolastatin conjugate complex of claim 1, wherein: the molar ratio of the anti-CD 56 antibody to the dolastatin is 1: 3.13.

9. The method for preparing the anti-CD 56 antibody and dolastatin coupled complex according to any one of claims 1 to 8, comprising the following steps:

a. preparing an anti-CD 56 antibody;

10. Use of the anti-CD 56 antibody-dolastatin conjugate complex of any one of claims 1-8 for the preparation of a medicament for the diagnosis and treatment of a tumor, immune system, or nervous system disorder.