CN105274116B

CN105274116B - Nucleic acid molecule for preparing humanized antibody and application thereof

Info

Publication number: CN105274116B
Application number: CN201510686616.3A
Authority: CN
Inventors: 刘作华; 葛良鹏; 刘雪芹; 邹贤刚; 丁玉春; 黄勇; 杨松全; 游小燕; 林保忠; 吴梦
Original assignee: Chongqing Camab Biotechnology Co ltd
Current assignee: Chongqing Jin Bo Bo Biotechnology Co., Ltd.
Priority date: 2015-10-21
Filing date: 2015-10-21
Publication date: 2020-09-29
Anticipated expiration: 2035-10-21
Also published as: WO2017067042A1; CN105274116A

Abstract

The invention provides a nucleic acid molecule, which comprises a human immunoglobulin gene or a fragment thereof, and is characterized by further comprising: human CD79 gene sequence. The invention overcomes the incompatibility problem of human immunoglobulin genes in different species due to the interaction of BCR with Ig alpha and Ig beta, and simultaneously, the expressed humanized antibody does not need to be reformed for the second time.

Description

Nucleic acid molecule for preparing humanized antibody and application thereof

Technical Field

The invention belongs to the technical field of biology, and mainly relates to a nucleic acid molecule and application thereof in preparation of a humanized antibody.

Background

Antibodies are an important class of biomedical products that play an important role in the prevention and treatment of human diseases. Therapeutic antibodies have evolved through different stages of development, murine, chimeric, reshaped, resurfaced, and fully humanized antibodies. A fully Humanized Antibody (Full Humanized Antibody) refers to an Antibody that has been genetically modified or immunized in a transgenic animal to obtain a protein sequence that is identical to that of a human Antibody. The fully humanized antibody does not contain animal protein, so that the side effect is low, the effect is better, and the fully humanized antibody becomes the main research and development direction of the current and future antibody engineering. Transgenic animal fully Humanized antibodies (Humanized antibodies) are obtained by transferring all or part of human immunoglobulin genes into the genome of an animal by Transgenic or artificial chromosome transfer techniques [ deletion (or inactivation) of endogenous Antibody genes in the animal ], so that the animal expresses the human antibodies.

The method is characterized in that the in vitro modification of human immunoglobulin genes is carried out, the human immunoglobulin genes are transferred into an animal body, and then the animal after the immune modification by the antigen is used for obtaining the antibody with high affinity, so that the important technology for researching and developing the humanized antibody is provided, and at present, 7 humanized antibodies prepared by the method are approved by the FDA in the United states. The mainstream method is to replace the animal immunoglobulin heavy chain and light chain genes with human immunoglobulin heavy chain and light chain genes, so that the human immunoglobulin heavy chain and light chain genes are rearranged in the animal body to generate human antibody protein. However, with the progress of research, it was found that the human immunoglobulin gene fragment can be rearranged and expressed in animals, but the performance of producing human antibody protein is lower than the production effect of animal autoantibody before gene modification. In the case of mice, the reason for this is mainly that when antibodies act as B cell surface receptors (BCRs) in the primary stage of B cell development, their interaction with murine signaling proteins Ig α and Ig β is not optimal (i.e., murine BCRs interact best with murine Ig α and Ig β or human BCRs with human Ig α and Ig β), thus affecting the class switching, affinity maturation and B cell development of antibodies into mature antibody-producing plasma cells. To overcome this problem, Lee et al (Nature biotechnology, 2014; 32 (4): 356-363) substitute the variable region of mouse immunoglobulin with the variable regions of three human immunoglobulin genes (IgH, Ig kappa and Ig lambda), and use the corresponding fragments of mouse immunoglobulin as the constant regions, and this strategy can overcome the above-mentioned problems to obtain chimeric antibodies with human variable regions and murine constant regions, and then transform the murine constant regions into human constant regions by downstream transformation to obtain fully humanized antibodies. This method has the disadvantage of requiring a second modification to obtain fully humanized antibodies.

Disclosure of Invention

The invention aims to provide a nucleic acid molecule capable of realizing rearrangement expression of humanized antibodies in animals, which overcomes the problem of incompatibility of human immunoglobulin genes in different species due to interaction of BCR with Ig alpha and Ig beta, and simultaneously, compared with the method adopted by Lee in the background art, the expressed humanized antibodies do not need to be subjected to secondary modification.

The invention provides a nucleic acid molecule, which comprises a human immunoglobulin gene or a fragment thereof and a human CD79 gene sequence.

The CD79 gene sequence includes CD79 alpha protein peptide and CD79 beta protein peptide coding sequence. The amino acid sequences of the CD79 alpha protein peptide and the CD79 beta protein peptide are shown as SED ID NO.3 and SED ID NO.2 respectively.

The human CD79 gene sequence is shown in SED ID NO. 1.

The transferred human CD79 sequence may be located on human immunoglobulin gene carrier or transferred into mouse or pig genome. Preferably, the human CD79 sequence is located at either end of a human Ig nucleic acid molecule. The human CD79 sequence is located at the front end of the V region or the back end of the C region of human immunoglobulin.

The structure of the CD79 gene sequence in the nucleic acid molecule is shown in FIG. 1-1. The position of CD79 in the above-described nucleic acid molecule is shown in FIGS. 1-2. The structure of the nucleic acid molecule is shown in FIGS. 1-3.

Preferably, the human immunoglobulin antibody gene or a fragment thereof is a human immunoglobulin antibody heavy chain gene. The present invention does not require internal modification of human immunoglobulin genes.

The human immunoglobulin gene includes a V region gene, a D region gene and a J region gene of a human immunoglobulin heavy chain gene. The human immunoglobulin heavy chain gene may also include hC μ, hC γ, hC α, hC and/or hC ξ heavy chain gene and/or human 3' regulatory region (hLCR), etc.

For example, the gene cluster of the above nucleic acid molecule is shown in FIG. 2 (the black box position is the introduced human CD79 sequence).

Through the transformation, the nucleic acid molecule can form a signal transmitter for B cell development, and is favorable for directly converting IgM cells into IgG cells and maturing affinity.

An expression vector comprising the nucleic acid molecule described above. A cell comprising the nucleic acid molecule or the vector. An animal, such as a mouse, a pig, etc., comprises the above nucleic acid molecule. A humanized antibody is prepared by rearranging and coding the nucleic acid molecules.

The use of the above nucleic acid molecule or vector or cell in the preparation of a humanized antibody. The application of the nucleic acid molecule or the vector or the cell in preparing transgenic animals.

The human immunoglobulin gene after the transformation is transferred into an animal body to obtain a transgenic animal of the human immunoglobulin, or further hybridized with an animal which does not express the self immunoglobulin gene to obtain a genetically engineered animal which only expresses the human antibody protein. The antigen is used to immunize animals with transferred human immunoglobulin genes (heavy chain and light chain) to obtain fully humanized antibody. For example:

the method for preparing the transgenic animal by adopting the nucleic acid molecule or the vector or the cell comprises the following steps:

(1) obtaining the nucleic acid molecule;

(2) constructing the nucleic acid molecule into a vector;

(3) introducing a vector containing the nucleic acid molecule into a host cell (including stem cells, induced stem cells and somatic cells) or an embryo;

(4) implanting cells containing human Ig into embryos (chimera preparation) or somatic clones of host animals;

(5) breeding heterozygous and homozygous animals transformed with human Ig genes (including crossing with animals in which the host's endogenous immunoglobulin genes are inactivated).

The host animal is mammal such as mouse, rabbit, pig, cattle, sheep, chicken, horse, etc. The vector is artificial chromosome (such as yeast, bacteria), bacteriophage, plasmid, etc. The method for introducing the vector into the host cell comprises electroporation, virus infection, lipofection, microinjection and the like.

Advantageous effects

1. The fully humanized antibodies produced by the present invention have high affinity.

2. The present invention can utilize one transgenic animal strain to produce various antibodies of different types, and the present invention is applicable to a variety of animals.

3. The host self-immunoglobulin of the present invention is not expressed or is below the detection limit.

4. The gene rearrangement efficiency of the invention is high, and the rearrangement, mutation and utilization rate of the VDJC gene are consistent with those of a human body.

5. The invention directly produces the full-humanized antibody without secondary modification, and the in vivo expression level can reach the level of healthy adults.

6. The present invention does not require internal modification of human immunoglobulin genes.

Drawings

FIG. 1-1 Structure of human CD79 gene

FIG. 1-2 shows the structure of the human CD79 gene and the selection gene

FIGS. 1-3 are graphs of the results of the transformation of the human CD79 gene and the hIg position.

FIG. 2 Structure of the modified human immunoglobulin heavy chain gene cluster

FIG. 3 example 1 Structure of human immunoglobulin Kappa light chain Gene Cluster

FIG. 4 example 1 Structure of human immunoglobulin lambda light chain Gene Cluster

FIG. 5 example 1 mouse immunoglobulin heavy chain Gene Cluster

FIG. 6 example 1 Gene targeting of knockout mouse immunoglobulin heavy chain Gene

FIG. 7 example 1 mouse immunoglobulin light chain Gene Cluster

FIG. 8 example 1 Gene targeting of knockout mouse immunoglobulin light chain Gene

Detailed Description

The present invention is described in detail below with reference to specific examples, which are given for the purpose of further illustrating the invention and are not to be construed as limiting the scope of the invention, and the invention may be modified and adapted by those skilled in the art in light of the above disclosure.

Example 1

The method comprises the following steps of transferring the modified human immunoglobulin gene into a mouse body, and immunizing the mouse containing the human immunoglobulin gene to obtain a fully humanized antibody:

1. optimized modification of human immunoglobulin gene

1) Modification of human immunoglobulin heavy chain gene

The human CD79 gene was transferred to YAC or BAC vector containing human Ig by homologous recombination to construct human immunoglobulin heavy chain gene cluster as shown in FIG. 2 (black box position is introduced human CD79 gene sequence). Comprises a V region, a D region and a J region which are human immunoglobulin heavy chain genes in sequence, hIgHC mu, hIgHC gamma 3, hIgHC gamma 1, hIgHC alpha 1, hLCR and hCD 79. The internal structure of hCD79 is shown in figure 1-1, and the gene sequence is shown in SED ID NO. 1; the coding sequences of the CD79 alpha protein peptide and the CD79 beta protein peptide are included; the amino acid sequences of the CD79 alpha protein peptide and the CD79 beta protein peptide are shown as SED ID NO.3 and SED ID NO.2 respectively.

2) Modification of human immunoglobulin Kappa light chain gene

The human immunoglobulin kappa light chain gene includes all or part of the V region, J region, C region, and KDE region of the human immunoglobulin kappa light chain gene. The gene cluster is shown in FIG. 3.

3) Modification of human immunoglobulin lambda light chain gene

The human immunoglobulin lambda light chain gene includes all or part of the V region, J region and C region of the human immunoglobulin lambda light chain gene, and an enhancer structure is added to the ends. The gene cluster is shown in FIG. 4.

2. Cultivation of humanized antibody transgenic mice

1) Cultivation of mouse with human immunoglobulin heavy chain gene

The human immunoglobulin heavy chain gene constructed in the step 1) is transferred into a mouse body by utilizing the existing conventional transgenic technology. The transgenic mouse with the human immunoglobulin heavy chain obtained by double-standard screening of PCR detection and ELISA detection.

The PCR identifying primers used were:

PCR 1

For：TGCTTGGAACTGGATCAGGCAGTC

Rev：TTGCTTAACTCCACACCTGCTCCTG

PCR product size：329bp

PCR2

For：TTGAGGAGACTGTCCATCCTTCAC

Rev：GAGAGGGCATCTTGGTCTTCTTTC

PCR product size：471bp

the ELISA-identified antibodies used were: sigma (I1886) and sigma (A8792), with serum from healthy adults and healthy mice as controls.

2) Cultivation of mouse with transgenic human immunoglobulin kappa light chain gene

The human immunoglobulin kappa light chain gene constructed in 2) of step 1 was transferred into mice using an existing conventional transgenic technique. Transgenic mice with the human immunoglobulin kappa light chain obtained by double-standard screening were tested by PCR and ELISA.

The PCR identifying primers used were:

PCR 1：

FOR：TGCTCTGACCTCTGAGGACCTGTCTGTA

Rev：TTCAGGCAGGCTCTTACCAGGACTCA

PCR product size：616bp

PCR 2：

For：CACCCAAGGGCAGAACTTTGTTACT

Rev：GAGGAAAGAGAGAAACCACAGGTGC

PCR product size：596bp

the ELISA-identified antibodies used were: sigma (K3502) and sigma (A7164), with sera from healthy adults and healthy mice as controls.

3) Cultivation of mouse with human immunoglobulin lambda light chain gene

The human immunoglobulin lambda light chain gene constructed in 3) of step 1 is transferred into a mouse body by using the existing conventional transgenic technology. Transgenic mice of human immunoglobulin lambda light chain obtained by double-standard screening of PCR detection and ELISA detection.

The PCR identifying primers used were:

PCR 1：

For：AGCACAATGCTGAGGATGTTGCTCC

Rev：ACTGACCCTGATCCTGACCCTACTGC

PCR product size：562bp

PCR 2：

FOR：CTCTGCTGCTCCTCACCCTCCTCACTCAGG

REV：GAGAGTGCTGCTGCTTGTATATGAGCTGCA

PCR product size：462bp

the ELISA-identified antibodies used were: sigma (L1645) and sigma (A5175), with healthy adult and healthy mouse sera as controls.

4) Cultivation of immunoglobulin heavy chain knockout mice (FIGS. 5 and 6)

An immunoglobulin heavy chain gene knockout mouse is constructed by using a Crispr/Cas9 technology. IgHC mu of the mouse immunoglobulin heavy chain gene is selected as a gene knockout site (the knockout site and the gene knockout effect are shown in figure 6), and the immunoglobulin heavy chain gene knockout mouse is obtained. And (3) screening the obtained immunoglobulin heavy chain gene knockout mice by using PCR detection and ELISA detection double standards.

The PCR identifying primers used were:

PCR：

For：AGCACCATTTCCTTCACCTGGAAC

Rev：CAAGGAGCAAATGACCATGTCTGG

PCR products: 760 bp. Then using BstEII enzyme to cut, the gene targeting is 753bp, and the gene targeting is two bands of 500bp and 260 bp.

The ELISA-identified antibodies used were: sigma (M8644) and sigma (A8786), with healthy adult and healthy mouse sera as controls.

5) Cultivation of immunoglobulin kappa light chain knockout mice (FIGS. 7 and 8)

The entire constant region (C) of the immunoglobulin Kappa light chain gene of the mouse was knocked out using a conventional gene knockout technique to obtain an immunoglobulin Kappa light chain gene knockout mouse. The mouse immunoglobulin kappa light chain gene knockout mouse is obtained by PCR detection and ELISA detection double-standard screening.

The PCR identifying primers used were:

PCR1：

For：CCCTTCCCTAGCCAAAGGCAACTA

Rev：CACAACGGGTTCTTCTGTTAGTCC

PCR product size：466

PCR 2：

For：CACACCTCCCCCTGAACCTGAAAC

Rev：GTTGTGGGTAGTGCCCAGCCTTGC

PCR product size：464bp

the ELISA-identified antibodies used were: southern Biotech (1170-01) and southern Biotech (1170-05) with healthy adult and healthy mouse sera as controls.

6) Hybrid combination to obtain humanized antibody transgenic mouse

Hybridizing and breeding the mice obtained in the second step 1), 2), 3), 4) and 5), and detecting by PCR and ELISA to finally obtain the humanized antibody transgenic mice which express high-level human immunoglobulin but do not express (or express low-level) mouse immunoglobulin for the next step of research.

3. Obtaining fully humanized antibodies

1) OVA immunization of humanized antibody transgenic mice obtained in step 2 and 6)

Mice 8 weeks old were selected for immunization.

Initial immune:

[ 1 ] OVA (Sigma A7641) antigen was diluted with PBS to a final concentration of 5mg/ml, 50ug CpG (ODN1826, tlrl-1826, Invivogen) was added, and an appropriate amount of aluminum hydroxide (vac-alu-50, Invivogen) was added to make the aluminum hydroxide concentration 1%.

② 0.75mL of antigen prepared in ① was mixed with CFA adjuvant (Sigma F5881) at a ratio of 1: 1 and mixed with MIXPAC^TMThe mice were immunized subcutaneously with 200ul (0.5mg) of the emulsion injected by syringe.

And (2) avoiding:

second immunization 16 days after priming, antigen was diluted with PBS to a final concentration of 2.5mg/ml, 25ug CpG was added, and an appropriate amount of aluminum hydroxide was added to make the concentration of aluminum hydroxide 1%.

② mixing ① prepared antigen 0.75mL with IFA adjuvant at a ratio of 1: 1, and mixing with MIXPAC^TMThe mice were immunized by intraperitoneal injection with 200ul (0.25mg) per injection.

And (3) three-step (I):

after the 16 th day of the secondary immunization, the tertiary immunization was performed, the antigen was diluted with PBS to a final concentration of 1.25mg/ml, 12.5ug of CpG was added, and an appropriate amount of aluminum hydroxide was added to make the concentration of aluminum hydroxide 1%.

② directly injecting antigen protein, preparing according to the method in (I), injecting 200ul (0.25mg) per mouse for intraperitoneal injection immunization.

2) Mouse antibody detection

10 days after 3 th immunization, mice No. 4115, 4116 and 4117 are respectively bled for ELISA detection, healthy adults and wild-type mice are used as controls, and the content of mouse IgG and the content of human IgG in the serum of the immunized mice are respectively detected, and the results are as follows:

firstly, detecting the content of mouse IgG

The kit is a mouse IgG ELISA kit (Abcam, ab157719)

4115: below detection limit

4116: below detection limit

4117: below detection limit

Healthy adults: below detection limit

Wild-type mice: 0.8mg/ml

The results show that: the humanized antibody mouse after immunization has extremely low expression level of the murine IgG.

② detection of human IgG in mouse serum

Human IgG ELISA kit (Abcam, ab100547) was used as the kit

4115：9.8mg/ml

4116：8.7mg/ml

4117：7.5mg/ml

Healthy adults: 10.2mg/ml

Wild-type mice: < 0.1ng/ml

The results show that: the humanized antibody mouse after immunization has high human IgG expression level.

③ determination of OVA antibody affinity

Selecting a 3556 mouse, fusing spleen cells and hybridoma cells, screening monoclonal antibodies, screening 3 cell clones with the highest expression quantity by ELISA, and performing anti-OVA antibody affinity detection by using a competitive ELISA detection method (CEB459Ge, Cloud-Clone Corp), wherein the result shows that the antibody with the highest affinity is 280 pM.

Example 2

The following results were obtained by transferring the modified human immunoglobulin gene into a pig as a host animal (immunoglobulin gene-knockout pig):

and (3) detecting the expression of human IgG in the pig blood: transgenic pigs with PCR detection positive 205 and 206 and transferred with the genes are selected, blood is collected, serum is separated, and the expression quantity of human IgG in the blood is detected, wherein healthy adults and ordinary pigs of the same month age are used as controls. The ELISA-identified antibodies used were: sigma (I1886) and sigma (A8792).

The results are as follows:

4115：0.5mg/ml

4116：0.1mg/ml

4117：0.2mg/ml

healthy adults: 10.2mg/ml

And (3) common pigs: < 0.1ng/ml

The results show that: the transferred gene can express human antibody heavy chain protein in pig body.

The scheme for modifying the human immunoglobulin gene is also applicable to other mammals, for example, pigs, rabbits, sheep, horses and the like are used as host animals, and the beneficial effect of the expression of the fully humanized antibody can be realized.

Claims

1. A nucleic acid molecule comprising a human immunoglobulin gene, further comprising: human CD79 gene sequence; the structure of the CD79 gene sequentially comprises a CD79 beta promoter, a CD79 beta sequence, a CD79 alpha sequence and a polyA signal; the human CD79 gene is shown in SEDIDNO.1.

2. The nucleic acid molecule of claim 1, wherein the CD79 α protein peptide is represented by SEDIDNO.3, and the CD79 β protein peptide is represented by SEDIDNO.2.

3. The nucleic acid molecule of claim 1 or 2, wherein the human CD79 gene is located at either end of the nucleic acid molecule.

4. The nucleic acid molecule of claim 1, wherein the human immunoglobulin gene comprises a V region gene, a D region gene, a J region gene of IgH.

5. The nucleic acid molecule of claim 1, wherein the human immunoglobulin heavy chain gene further comprises an hC γ, hC α, hC, and/or hC ξ heavy chain gene and/or hLCR.

6. The nucleic acid molecule of claim 1, wherein the human CD79 gene is located at the front end of a V region or the rear end of a C region of a human immunoglobulin.

7. A vector comprising the nucleic acid molecule of any of claims 1-6.

8. Use of a nucleic acid molecule according to any one of claims 1 to 6 or a vector according to claim 7 for the preparation of a transgenic animal.

9. A method for producing a transgenic animal using the nucleic acid molecule of any one of claims 1 to 6 or the vector of claim 7, comprising the steps of:

(1) obtaining the nucleic acid molecule;

(2) constructing the nucleic acid molecule into a vector;

(3) introducing a vector containing the nucleic acid molecule into a host cell or embryo;

(4) implanting cells containing human Ig into embryo or somatic cell clone of host animal;

(5) breeding heterozygous and homozygous animals transformed with human Ig gene.