CN114907480A - Anti-human CD73 monoclonal antibody without hook effect - Google Patents

Abstract

The invention provides a brand-new anti-CD 73 monoclonal antibody. The antibody has high affinity with human CD73 protein; biochemical level and cell level experiments show that the compound has high-efficiency CD73 enzyme activity inhibition effect; in vivo experiments show that the antibody has the function of obviously inhibiting the growth of tumor cells. In particular, the antibody does not generate the 'hook effect' of the inhibition effect of most antibodies in the prior art on the enzyme activity of CD73, and is more suitable for clinical application.

Description

Anti-human CD73 monoclonal antibody without hook effect

The technical field is as follows:

the invention relates to a genetic engineering antibody, in particular to an anti-human CD73 monoclonal antibody with a brand-new sequence and no hook effect.

Background art:

CD73 (ecto-5' -nucleotidase), a multifunctional extracellular nuclease, is highly expressed in most solid tumors, and CD73 promotes the proliferation of tumor cells and is closely related to the staging, pathological type, and prognosis outcome of tumors.

Most of the CD73 antibodies are confronted with the "HOOK effect" (HOOK effect), that is, the therapeutic effect of the antibodies begins to increase negatively with the increase of the antibody concentration after reaching the peak value with the change of the dose-effect relationship of the antigen and the antibody. Therefore, the optimal dosage of the antibody for different individuals is difficult to determine, resulting in poor actual therapeutic effect. Such as the therapeutic CD73 monoclonal antibody MEDI-9447.

Thus, an anti-human CD73 antibody free of hook effect can be obtained, which can provide an anti-tumor agent more suitable for clinical use.

Disclosure of Invention

The invention aims to provide a monoclonal antibody capable of effectively inhibiting the activity of CD73, which is used for preparing a medicament for treating tumor-related diseases.

The invention uses human CD73 protein to immunize BABL/C mouse, and obtains brand-new anti-human CD73 monoclonal antibody through hybridoma screening; the monoclonal antibodies are IgG types.

The heavy chain and light chain sequences of the anti-human CD73 monoclonal antibody obtained by the invention are completely different from those of the anti-human CD73 monoclonal antibody in the prior art;

the human CD73 protein used in the invention is a human-derived CD73 protein autonomously expressed by the applicant, and the mouse used is a BABL/C mouse.

Specifically, the present invention accomplishes the above-mentioned work by the following means:

a human CD73 protein as antigen was immunized at 30. mu.g/mouse BABL/C, three weeks later the same dose boosted;

b, measuring the antibody titer in the serum of the immunized mouse by an ELISA method, and performing impact immunization at a dose of 50 mu g after an ideal effect is achieved;

c, fusing the spleen cells of the mice successfully immunized with SP2/0 cells, carrying out supernatant titer detection after the cells grow into cell masses, and obtaining a positive monoclonal cell strain through three rounds of subcloning;

d, carrying out mouse intraperitoneal injection to prepare ascites after the monoclonal cell strain is subjected to amplification culture, and purifying the collected ascites to obtain a corresponding antibody;

e, utilizing an SPR technology to carry out affinity kinetic determination on the monoclonal antibody;

f, measuring the inhibition effect of the monoclonal antibody on the enzymatic activity of the CD 73;

g measures the tumor suppression effect of the monoclonal antibodies on the transplanted tumor model.

The monoclonal antibody of anti-human CD73 obtained by the invention is named as 7-C10-Ba-C2. The molecular basis for the specificity of this antibody is mainly derived from the highly variable regions CDR1, CDR2 and CDR3 of its heavy and light chains, which are key sites for antigen binding.

The CDR1, CDR2 and CDR3 of the heavy chain and the light chain of the monoclonal anti-human CD73, which are obtained by the invention, are respectively polypeptides with amino acid sequences shown as follows:

antibody 7-C10-Ba-C2:

heavy chain CDR 1: SEQ ID No. 1; heavy chain CDR 2: SEQ ID No. 2; heavy chain CDR 3: SEQ ID No. 3;

light chain CDR 1: SEQ ID No. 4; light chain CDR 2: SEQ ID No. 5; light chain CDR 3: SEQ ID No. 6;

the term "monoclonal antibody" as used herein should be construed to encompass any specific binding member having a binding domain of the desired specificity, either monovalent or single chain antibodies, diabodies, chimeric antibodies, as well as derivatives, functional equivalents and homologs of any of the foregoing, and also includes antibody fragments and any polypeptides comprising an antigen binding domain.

Examples of the monoclonal antibody of the present invention are an immunoglobulin IgG subtype and a subtype subclass thereof;

although the molecular basis for antibody specificity arises primarily from its heavy and light chain hypervariable regions CDR1, CDR2, and CDR3, the sequences of the CDRs should be retained as much as possible in order to maintain preferred binding properties. However, although it is possible to achieve the object of the invention, even more optimal binding properties, if there are individual amino acid changes. However, individual amino acid changes do not depart from the spirit and concept of the present invention.

In addition to the highly variable regions CDR1, CDR2, and CDR3 in the heavy and light chains as described above, the others are framework regions. The framework regions may be replaced by other sequences under conditions that do not affect the desired three-dimensional structure of the binding.

The invention has the beneficial effects that:

experiments prove that the anti-human CD73 monoclonal antibody produced by the invention has the following outstanding characteristics:

1. affinity kinetics experiments show high affinity to human CD73 (see example 2 for details)

2. The biochemical level experiment and the cell level experiment both show that the antibody has high-efficiency CD73 enzyme activity inhibition effect, and different from most CD73 antibodies reported at present, the anti-human CD73 antibody has no hook effect on the inhibition effect of CD73 enzyme activity (see example 4 for details)

3. Pharmacodynamic evaluation experiments in animals show that the growth of the transplanted tumor of the human immune reconstituted mouse can be obviously inhibited (see example 5 for details).

Drawings

FIGS. 1 and 2 show the results of the affinity kinetic analysis of example 2, wherein Ka, Kd and KD are the binding constant, dissociation constant and affinity constant, respectively.

Wherein, FIG. 1 shows the experimental results of the anti-CD 73 monoclonal antibody (7-C10-Ba-C2) of the present invention;

FIG. 2 shows the results of the test with the control antibody Contbody (anti-CD 73 monoclonal antibody MEDI-9447 from MedImmune).

FIG. 3 shows the results of experiments on the inhibition of the enzyme activity of CD73 by biochemical level antibodies in example 3 (inhibition of the enzyme activity of CD73 by 7-C10-Ba-C2 monoclonal antibody);

FIG. 4 shows the results of the experiments in example 3 (inhibition of CD73 enzyme activity by 7-C10-Ba-C2 monoclonal antibody) in which the cell-level (A549) antibody inhibits CD73 enzyme activity

FIG. 5 shows the results of the experiment in example 4 (evaluation of the animal drug efficacy of 7-C10-Ba-C2 monoclonal antibody)

The above-mentioned 7-C10-Ba-C2 antibody is the name of the anti-human CD73 monoclonal antibody obtained by the present invention.

Sequence information:

SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, CDR1, CDR2 and CDR3 of the heavy chain variable region of anti-human CD73 monoclonal antibody 7-C10-Ba-C2, respectively;

SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, CDR1, CDR2 and CDR3 of the light chain variable region of anti-human CD73 monoclonal antibody 7-C10-Ba-C2, respectively;

SEQ ID NO.7 and SEQ ID NO.8 are amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-human CD73 monoclonal antibody 7-C10-Ba-C2, respectively.

Detailed Description

In order to make the objects, technical solutions and effects of the present invention clearer and clearer, the following examples further illustrate the present invention in detail. It should be understood that the specific methods, reagents, etc. used in the examples are illustrative only and are not limiting upon the scope of the invention.

The present invention provides the heavy and light chain sequences of a specific anti-human CD73 monoclonal antibody. The monoclonal antibody is expressed by a corresponding monoclonal cell strain obtained by screening hybridomas after a BABL/C mouse is immunized by human CD73 protein; the monoclonal antibodies are IgG types.

The antigen used in the following examples is an autonomously expressed human CD73 protein, the C-terminus of which comprises a 6 × His tag;

the used immunologic adjuvant is a 5-week rapid immunologic adjuvant (cargo number: KX0210041) of Beijing Boolong immunization technology Limited company, the immunization is strengthened once after 21 days of primary immunization, and the cell fusion can be carried out once by using antigen impact immunization before the cell fusion;

the fusion method is electrofusion, the electrofusion instrument is ECM2001 of BTX, and the fusion buffer solution is original factory cell fusion solution (Cat: 47-0001);

after growing cell masses, fused cells adopt ELISA to detect the expression of a supernatant antibody, an ELISA plate coats human CD73 protein or His protein, and the His protein is used for eliminating false positive holes of Anti-His;

carrying out subcloning on the positive hole by adopting a limiting dilution method, carrying out three rounds of subcloning, and finally obtaining a positive monoclonal cell strain;

and (3) preparing the positive monoclonal antibody, purifying the ascites to obtain a corresponding monoclonal antibody, and further using the monoclonal antibody for affinity determination, CD73 enzyme activity inhibition experiments and animal efficacy evaluation.

The "7-C10-Ba-C2" described in the following examples is the name of the anti-human CD73 monoclonal antibody provided by the present invention.

Example 1 antigen immunization, cell fusion and screening of Positive clones and preparation and purification of ascites antibodies

The purpose of the experiment is as follows:

monoclonal antibodies were prepared using the autonomously expressed human CD73 protein as an antigen.

The experimental method comprises the following steps:

anti-human CD73 monoclonal antibodies were prepared using hybridoma technology. The specific method comprises the following steps:

female BABL/C mice at 4-6 weeks were immunized with 30 μ g of human CD73 protein.

The same method was used to boost once on day 21 after the first immunization.

Serum titers were measured by ELISA from canthal bleeds at day 35 after the first immunization.

After the antibody titer reaches the requirement, 50 mu g of human CD73 protein is adopted for antigen impact immunization.

Splenocytes taken 3 days after the impact immunization were electrofused with SP2/0 cells, and the titers of anti-human CD73 antibodies in the hybridoma supernatants were detected by ELISA after the cells had clumped.

The experimental results are as follows:

through three rounds of subcloning, and through double screening of affinity and CD73 enzyme activity inhibition, a monoclonal cell strain with high expression of the anti-human CD73 antibody is finally obtained and named as 7-C10-Ba-C2. Amplifying the monoclonal cells, and then preparing a purified antibody from ascites for subsequent affinity determination, CD73 enzyme activity inhibition experiments and animal drug effect determination.

The obtained monoclonal antibody has high affinity, can effectively inhibit the enzyme activity of CD73 without hook effect, and simultaneously, the animal drug effect shows an ideal tumor inhibition effect.

Example 27-C10-Ba-C2 monoclonal antibody and recombinant human CD73 affinity kinetic analysis

Purpose of the experiment:

the affinity kinetic constants of the 7-C10-Ba-C2 monoclonal antibody and the control antibody were measured using the Biacore T200 system.

Reagents and methods:

the Mouse Antibody Capture Kit is a commercial Kit purchased from GE company, an anti-Mouse Fc IgG is fixed on a CM5 sensor chip by adopting an amino coupling method, a detected Antibody is captured by the coupled anti-Mouse Fc IgG, then a series of human CD73 proteins with concentration gradient are injected, and a Glycine-Hcl regeneration detection chip with the pH value of 1.7 of the Kit is adopted after sample injection is finished.

The running buffer was HBS-EP + (10mM HEPES, pH7.4, 150mM NaCl, 3mM EDTA and 0.05% P20), measured at 25 ℃;

the control substance selected in the experiment is MEDI-9447; MEDI-9447 is an anti-CD 73 monoclonal antibody from MedImmune, which was synthesized and purified according to the sequence of the patent (US2016/0194407A1) to obtain a control and named as Contbody.

Using Biacore T200 evaluation software, according to 1: 1 binding model the data were fitted and the binding (Ka) and dissociation (Kd) rate constants and equilibrium constants (Kd) were calculated.

The experimental results are as follows:

according to the detection results, the data of the affinity results of the present and control antibodies are shown in fig. 1, fig. 2 and the following table:

experimental antibodies	Ka(1/Ms)	Kd(1/s)	KD(M)
				Antibodies of the invention 7-C10-Ba-C2	7.721×10 4	3.214×10 -5	4.162×10 -10
Control antibody MEDI-9447	2.785×10 5	3.912×10 -5	1.405×10 -10

The results show that the 7-C10-Ba-C2 monoclonal antibody has high affinity with recombinant human CD73, and the affinity is equivalent to that of a control.

And (4) experimental conclusion:

the 7-C10-Ba-C2 monoclonal antibody obtained by the invention has high affinity with human CD 73.

Example 37-inhibition of the enzymatic Activity of CD73 by the C10-Ba-C2 monoclonal antibody

Purpose of the experiment:

the ability of the 7-C10-Ba-C2 monoclonal antibody to inhibit the activity of CD73 enzyme was examined at the biochemical level and the cellular level, respectively.

The experimental method comprises the following steps:

1. biochemical method

The 7-C10-Ba-C2 monoclonal antibody and the control MEDI-9447(Contbody) were added to the bottom of a 96-well white plate in a suitable concentration gradient, then CD73 protein was added to each well to a final concentration of 0.25. mu.g/mL, and the plate was incubated at 37 ℃ for 15min, after the incubation was completed, AMP and ATP were added to final concentrations of 500. mu. mol/L and 100. mu. mol/L, respectively, and the plate was incubated at 37 ℃ for 30 min. Finally, an equal volume of Cell titer Glo was added to each well.

Detecting the signal value of each hole by a chemiluminescence method of an enzyme-labeling instrument, and calculating and processing data;

2. cell method

The 7-C10-Ba-C2 monoclonal antibody and the control MEDI-9447 (contact) were added to the bottom of the 96-well plate at an appropriate concentration gradient, and the A549 cells were digested, resuspended, counted, and counted at 1X 10 ⁵ Inoculating the density of each hole to the corresponding hole, incubating in a carbon dioxide incubator for 15min, adding AMP to each hole after incubation is finished, wherein the final concentration is 500 mu mol/L, and continuously incubating in the carbon dioxide incubator for 24 h; after 24h, the 96-well plate is taken out and centrifuged at 1000rpm for 5min, 50. mu.L of supernatant is taken out of each well to the corresponding position of a new 96-well white plate, then an ATP solution is added into each well to the final concentration of 100. mu. mol/L, and then equal volume of Cell titer Glo is added into each well.

And (3) detecting the signal value of each hole by chemiluminescence of the microplate reader, and calculating and processing data.

The experimental results are as follows: see fig. 3 and 4

The inhibitory effect of the enzyme activity of each antibody CD73, EC50, is summarized in the following table, in which the control is MEDI-9447 (contact):

7-C10-Ba-C2 has the same inhibition effect on the enzyme activity of CD73 at the biochemical level and the cellular level as the control MEDI-9447 (contact); the inhibition of MEDI-9447 on enzyme activity begins to gradually decrease with the increase of antibody concentration, and the enzyme activity curve shows a hook shape (hook effect). The inhibition of the enzyme activity of the CD73 by the 7-C10-Ba-C2 is always maintained at a high level along with the increase of the antibody concentration and does not show a hook effect, which is different from the control MEDI-9447;

and (4) experimental conclusion:

the biochemical level experiment and the cell level experiment show that the anti-CD 73 monoclonal antibody obtained by the invention has high-efficiency CD73 enzyme activity inhibition effect.

Example 47-evaluation of animal drug efficacy of C10-Ba-C2 monoclonal antibody

Purpose of the experiment: in vivo experiments tested the effect of the 7-C10-Ba-C2 monoclonal antibody on inhibiting the growth of tumor cells.

The experimental method comprises the following steps:

90B-NDG mice were taken and acclimatized for at least one week.

Culturing A375 cells, transferring every other day for one passage, collecting cells, and adjusting the final concentration of cells to 5 x 10 with PBS ⁷ Each 0.1mL of the cells was inoculated subcutaneously into the right shoulder of the mouse.

Selecting tumor with volume of 20-30mm about 10 days after inoculation ³ The mice were divided into groups of 10 mice each, for a total of 3 groups.

PBMC cells were revived on the day of grouping, PBS adjusted cell concentration to 25million/mL, and each mouse was i.v. injected with 200 μ L (5 million) PBMC, then i.v. dosed, and tumor volumes were measured 2 times per week post dose; the frequency of administration was Q3D for 10 times.

Drawing tumor growth curves of each group by taking the tumor volume as a vertical coordinate and taking the administration time as a horizontal coordinate; performing one way ANOVA analysis on each drug group and a control group respectively, and comparing the difference between the two groups, wherein p is less than 0.05; p < 0.01; p < 0.001.

The experimental results are as follows: see fig. 5. The experimental result shows that compared with the control group IgG, the 7-C10-Ba-C2 monoclonal antibody shows a remarkable effect of inhibiting the growth of tumor cells.

And (4) experimental conclusion:

the anti-CD 73 monoclonal antibody 7-C10-Ba-C2 has the function of obviously inhibiting the growth of tumor cells.

Sequence listing

<110> New medicine Co., Ltd, Jiangsu province

<120> anti-human CD73 monoclonal antibody without hook effect

<160> 8

<170> PatentIn version 3.1

<210> 1

<211> 5

<212> PRT

<213> mouse (Mus musculus)

<400> 1

Gly Tyr Thr Met Asn

1 5

<210> 2

<211> 17

<212> PRT

<213> mouse (Mus musculus)

<400> 2

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

1 5 10 15

<210> 3

<211> 10

<212> PRT

<213> mouse (Mus musculus)

<400> 3

Glu Gly Tyr Asp Gly Gly Tyr Phe Asp Tyr

1 5 10

<210> 4

<211> 11

<212> PRT

<213> mouse (Mus musculus)

<400> 4

Arg Ala Ser Glu Asn Ile Tyr Ser Tyr Leu Ala

1 5 10

<210> 5

<211> 7

<212> PRT

<213> mouse (Mus musculus)

<400> 5

Asn Thr Gln Thr Leu Ala Glu

1 5

<210> 6

<211> 10

<212> PRT

<213> mouse (Mus musculus)

<400> 6

Gln His His Tyr Gly Thr Pro Met Tyr Pro

1 5 10

<210> 7

<211> 119

<212> PRT

<213> mouse (Mus musculus)

<400> 7

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

1 5 10 15

Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Glu Gly Tyr Asp Gly Gly Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser

115

<210> 8

<211> 108

<212> PRT

<213> mouse (Mus musculus)

<400> 8

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

1 5 10 15

Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val

35 40 45

Tyr Asn Thr Gln Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Met

85 90 95

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

100 105

Claims (10)

1. The application of one or two groups of the following two groups of polypeptides in preparing anti-human CD73 monoclonal antibodies, wherein each group consists of three polypeptides, and the amino acid sequences are shown as follows:

SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3;

SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6.

2. The use of one or both of the polypeptides of claim 1 in the preparation of an anti-tumor medicament.

3. A monoclonal antibody comprising one or both of the polypeptides of claim 1.

4. An anti-human CD73 monoclonal antibody, wherein the CDR1, CDR2 and CDR3 in the heavy chain variable region are respectively the following amino acid sequence polypeptides: SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3; the CDR1, CDR2 and CDR3 in the light chain variable region are respectively the following amino acid sequence polypeptides: SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6.

5. The use of the monoclonal antibody of claim 4 in the preparation of inhibitors of CD73 enzyme activity or anti-tumor drugs.

6. An antitumor agent comprising the monoclonal antibody according to claim 4.

7. An inhibitor of CD73 enzyme activity comprising the monoclonal antibody of claim 4.

8. A polypeptide of the amino acid sequence: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6.

9. Use of one or more of the polypeptides of claim 8 for the preparation of an anti-human CD73 monoclonal antibody.

10. The use of one or more of the polypeptides of claim 8 in the preparation of inhibitors of CD73 enzyme activity or anti-tumor drugs.

Images