CN114907480B

CN114907480B - Anti-human CD73 monoclonal antibodies without hook effect

Info

Publication number: CN114907480B
Application number: CN202210460719.8A
Authority: CN
Inventors: 廖高勇; 丁海剑; 张怡
Original assignee: Xintrum Pharmaceuticals Ltd
Current assignee: Xintrum Pharmaceuticals Ltd
Priority date: 2022-04-28
Filing date: 2022-04-28
Publication date: 2023-06-13
Anticipated expiration: 2042-04-28
Also published as: CN114907480A

Abstract

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

Description

Anti-human CD73 monoclonal antibodies without hook effect

Technical field:

the invention relates to a genetic engineering antibody, in particular to a novel sequence anti-human CD73 monoclonal antibody without hook effect.

The background technology is as follows:

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

Most CD73 antibodies currently suffer from the "HOOK effect" (HOOK effect), i.e. as the quantitative relationship of the antigen and antibody changes, the therapeutic effect of the antibody begins to increase negatively with increasing antibody concentration after peaking. Thus, the optimal dosage of antibody for different types of individuals is difficult to determine, resulting in poor actual therapeutic effect. Such as therapeutic CD73 monoclonal antibody MEDI-9447.

Thus, obtaining an anti-human CD73 antibody without a hook effect may provide an anti-tumor formulation 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 medicines for treating tumor-related diseases.

According to the invention, after a BABL/C mouse is immunized by human CD73 protein, a brand-new anti-human CD73 monoclonal antibody is obtained through hybridoma screening; the monoclonal antibody type is of the IgG type.

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 human CD73 protein which is expressed by the applicant autonomously, and the used mice are BABL/C mice.

Specifically, the invention accomplishes the above-mentioned work by:

human CD73 protein A is used as antigen to immunize BABL/C mice at 30 mug/mouse, and the same dose is used for boosting after three weeks;

b ELISA method for determining antibody titer in serum of immunized mice, and after reaching ideal effect, the mice are immunized by impact at 50 mug dosage;

c, taking splenocytes of mice which are successfully immunized and fusing with SP2/0 cells, detecting the titer of the supernatant after the cells grow into cell clusters, and obtaining positive monoclonal cell strains through three rounds of subcloning;

d, performing intraperitoneal injection on the mice after the expanded culture of the monoclonal cell strain to prepare ascites, and purifying the collected ascites to obtain a corresponding antibody;

e, carrying out affinity kinetics measurement of the monoclonal antibody by utilizing an SPR technology;

f, determining the inhibition effect of the monoclonal antibody on the activity of the CD73 enzyme;

g the tumor inhibition of the transplanted tumor model by the monoclonal antibody was measured.

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

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

antibody 7-C10-Ba-C2:

heavy chain CDR1: SEQ ID NO.1; heavy chain CDR2: SEQ ID NO.2; heavy chain CDR3: SEQ ID NO.3;

light chain CDR1: SEQ ID NO.4; light chain CDR2: SEQ ID NO.5; light chain CDR3: SEQ ID NO.6;

the term "monoclonal antibody" as used herein is to be construed as encompassing any specific binding factor having a binding domain of the desired specificity, and may be monovalent or single chain antibodies, diabodies, chimeric antibodies, and derivatives, functional equivalents and homologs of the above antibodies, as well as antibody fragments and any polypeptide comprising an antigen binding domain.

Examples of monoclonal antibodies of the invention are immunoglobulin IgG subtypes and subclasses thereof;

although the molecular basis of antibody specificity is primarily derived from its heavy and light chain highly variable regions CDR1, CDR2 and CDR3, the sequences of these CDRs should be as preserved as possible in order to maintain the preferred binding properties. However, although individual amino acid changes, it is possible to achieve the object of the invention and even optimize the binding properties. However, individual amino acid changes do not depart from the spirit and scope 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 with other sequences without affecting the three-dimensional structure required for 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 kinetic experiments showed high affinity for human CD73 (see example 2 for details)

2. Both biochemical and cellular level experiments showed a highly potent inhibition of CD73 enzyme activity and, unlike most of the CD73 antibodies reported so far, the anti-human CD73 antibodies of the present invention have no hook effect on inhibition of CD73 enzyme activity (see example 4 for details)

3. The in vivo pharmacodynamics evaluation experiment of animals shows that the growth of human immune reconstruction mouse transplantation tumor can be obviously inhibited (see the embodiment 5 for details).

Drawings

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

Wherein FIG. 1 is the experimental result of the anti-CD 73 monoclonal antibody (7-C10-Ba-C2) of the invention;

FIG. 2 shows the experimental results of a control antibody Contbody (anti-CD 73 monoclonal antibody MEDI-9447 of MedImmune Co.).

FIG. 3 is the experimental results of the inhibition of CD73 enzyme activity by the biochemical level antibodies of example 3 (inhibition of CD73 enzyme activity by 7-C10-Ba-C2 monoclonal antibodies);

FIG. 4 is a graph showing the results of an experiment of the inhibition of CD73 enzyme activity by the cell level (A549) antibody of example 3 (inhibition of CD73 enzyme activity by the 7-C10-Ba-C2 monoclonal antibody)

FIG. 5 shows the results of experiments in example 4 (evaluation of animal 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 are CDR1, CDR2 and CDR3, respectively, of the heavy chain variable region of the anti-human CD73 monoclonal antibody 7-C10-Ba-C2;

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

SEQ ID No.7 and SEQ ID No.8 are the 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

The following examples are provided to illustrate the present invention in further detail in order to make the objects, technical solutions and effects of the present invention more clear and clarified. It should be understood that the specific methods, reagents, etc. used in the examples are illustrative of the present invention and are not intended to limit the scope of the invention.

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

The antigen used in the following examples was an autonomously expressed human CD73 protein, the C-terminus of which contained a 6 xhis tag;

the used immune adjuvant is 5-week rapid immune adjuvant (product number: KX 0210041) of Beijing Boolone immune technology Co., ltd, and is boosted once after 21 days of primary immunization, and the antigen is used for carrying out primary impact immunization before cell fusion to carry out cell fusion;

the fusion method is an electrofusion method, the electrofusion instrument is ECM2001 type of BTX company, and the fusion buffer solution is cell fusion solution of a primary factory (product number: 47-0001);

after growing cell clusters, the fused cells adopt ELISA to detect the expression of 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;

subcloning the positive hole by adopting a limiting dilution method, and subcloning for three times to finally obtain a positive monoclonal cell strain;

and (3) carrying out ascites preparation on the positive monoclonal antibody, purifying 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 examples below is the designation of the anti-human CD73 monoclonal antibodies provided herein.

EXAMPLE 1 antigen immunization, cell fusion, screening of Positive clones 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 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 were immunized with 30 μg of human CD73 protein for 4-6 weeks.

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

Serum was isolated by canthus blood sampling at day 35 after the first immunization and assayed by ELISA for serum titer.

After the antibody titer reached the requirement, antigen challenge immunization was performed with 50 μg of human CD73 protein.

Spleen cells were electrofused with SP2/0 cells 3 days after impact immunization, and ELISA was used to detect anti-human CD73 antibody titers in hybridoma supernatants after cell clumping.

Experimental results:

through three rounds of subcloning and double screening of affinity and CD73 enzyme activity inhibition, the anti-human CD73 antibody high-expression monoclonal cell strain is finally obtained and named 7-C10-Ba-C2. And (3) carrying out ascites preparation and purification on the monoclonal cells after amplification, and using the monoclonal cells in subsequent affinity determination, CD73 enzyme activity inhibition experiments and animal drug effect determination.

Proved by verification, the obtained monoclonal antibody has high affinity, can effectively inhibit the activity of CD73 enzyme and has no hook effect, and meanwhile, the animal drug effect shows ideal tumor inhibiting effect.

EXAMPLE 2 7-C10-Ba-C2 monoclonal antibodies and recombinant human CD73 affinity kinetic analysis

The purpose of the experiment is as follows:

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

Reagents and methods:

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

The running buffer was HBS-EP+ (10mM HEPES,pH7.4, 150mM Nacl,3mM EDTA and 0.05% P20) and the assay temperature was 25 ℃;

the control selected in the experiment was MEDI-9447; MEDI-9447 is an anti-CD 73 monoclonal antibody from MedImmune, which we expressed synthetically according to the sequence of its patent (US 2016/0194407A 1) and purified to give a control and named Contbody.

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

Experimental results:

based on the detection results, the affinity results data of the inventive and control antibodies are shown in fig. 1, fig. 2 and the following table:

experimental antibodies	Ka(1/Ms)	Kd(1/s)	KD(M)
				The antibody 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 showed that the 7-C10-Ba-C2 monoclonal antibody had high affinity for recombinant human CD73, which was comparable to the control.

Conclusion of experiment:

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

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

The purpose of the experiment is as follows:

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

The experimental method comprises the following steps:

1. biochemical method

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

Detecting signal values of all holes by using a chemiluminescent method of an enzyme-labeled instrument, and calculating and processing data;

2. cell method

The 7-C10-Ba-C2 monoclonal antibody and control MEDI-9447 (Contbody) were added to the bottom of 96-well plates at appropriate concentration gradients, counted 1X 10 after A549 cell digestion and resuspension ⁵ Inoculating density of each hole to a corresponding hole, incubating for 15min in a carbon dioxide incubator, adding AMP to each hole after incubation, and incubating in the carbon dioxide incubator for 24h, wherein final concentration is 500 mu mol/L; after 24h, the 96-well plates were spun down at 1000rpm for 5min, 50. Mu.L of supernatant was removed from each well to the new 96-Kong Baiban corresponding position, after which ATP solution was added to a final concentration of 100. Mu. Mol/L per well, and then an equal volume of Celltiter Glo was added per well.

And detecting signal values of all holes by using chemiluminescence of the enzyme-labeled instrument, and calculating and processing data.

Experimental results: see FIG. 3 and FIG. 4

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

the inhibition of CD73 enzyme activity by 7-C10-Ba-C2 at the level of biochemical and cellular levels is comparable to that of the control MEDI-9447 (Contbody); whereas inhibition of enzyme activity by MEDI-9447 begins to decrease gradually with increasing antibody concentration, the enzyme activity profile exhibits a hook-like (hook effect). Unlike the control MEDI-9447, inhibition of CD73 enzyme activity by 7-C10-Ba-C2 was maintained at a high level at all times with increasing antibody concentration, and did not exhibit a hook effect;

conclusion of experiment:

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

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

The purpose of the experiment is as follows: in vivo experiments test 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 housed adaptively for at least one week.

Culturing A375 cells, transferring for one generation every other day, collecting cells, and PBS (phosphate buffer solution) regulating final concentration to 5×10 ⁷ Each 0.1mL was inoculated subcutaneously into the right shoulder of the mice.

About 10 days after inoculation, selecting tumor volume of 20-30mm ³ Is grouped into 10 mice per group, 3 mice per group.

PBMC cells were resuscitated on the day of grouping, PBS was adjusted to a cell concentration of 25million/mL, 200 μl (5 million) PBMC were injected i.v. per mouse, and then i.v. was administered, after which tumor volumes were measured 2 times per week; the frequency of administration was Q3D for a total of 10 times.

Drawing tumor growth curves of each group by taking tumor volume as an ordinate and administration time as an abscissa; one way ANOVA analysis is carried out on each drug group and the control group respectively, and the difference between the two is compared, wherein p is less than 0.05; * P <0.01; * P <0.001.

Experimental results: see fig. 5. Experimental results show that compared with the control group IgG, the 7-C10-Ba-C2 monoclonal antibody has obvious effect of inhibiting the growth of tumor cells.

Conclusion of experiment:

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

Sequence listing

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Claims

1. An anti-human CD73 monoclonal antibody, characterized in that: CDR1, CDR2 and CDR3 in the heavy chain variable region are polypeptides of the amino acid sequences: SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3; CDR1, CDR2 and CDR3 in the light chain variable region thereof are polypeptides of the amino acid sequences: SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6.

2. The use of the monoclonal antibody of claim 1 for preparing an inhibitor of CD73 enzyme activity or for preparing an anti-melanoma drug.

3. An antitumor agent comprising the monoclonal antibody of claim 1.

4. A CD73 enzyme activity inhibitor comprising the monoclonal antibody of claim 1.