Patent Application
20250234846
This application claims the priority benefit of China application serial no. 202410082783.6, filed on Jan. 19, 2024. The entirety of each of the above-mentioned patent applications is hereby incorporated by reference herein and made a part of this specification.
The instant application contains a Sequencing Listing which has been submitted electronically in XML file and is hereby incorporated by reference in its entirety. Said XML copy, created on Jan. 16, 2025, is named 153347-US-SEQUENCING_LIST and is 10,982 bytes in size.
The present invention belongs to the field of biotechnology, and in particular relates to a method for preparing a genetically modified non-human mammal and use thereof.
In camelids and cartilaginous fish, a uniquely structured antibody, called heavy-chain-only antibody, occurs naturally. This antibody consists of only heavy chains and its variable region fragment has a molecular weight of 15 kDa, about one-tenth that of a conventional antibody which is 150-160 kDa. The variable region fragment of a heavy-chain-only antibody is also known as a nanobody. Nanobodies have been widely used in the development of bispecific/multispecific antibodies, in the CAR-T cell therapy, etc. As of January 2023, four nanobody-based therapeutics have been marketed, among which the nanobody-based BCMA CAR-T therapy developed by Legend Bio has achieved excellent clinical results. In addition, more than 10 nanobody molecules developed as neutralizing antibodies have entered clinical phase I1/III. The drug development and application of nanobodies are still in a relatively early stage, which shows great application potential and development prospects.
Fully human nanobody-producing mice are useful for the development of nanobody drugs. Based on the independently developed humanization technology for genome fragments at a level ranging from hundreds of kilobase pairs to mega base pairs (abbreviated as MB), humanization of heavy chain genes of antibodies, encompassing the major human heavy chain variable region genes, has been achieved. HuNano Mouse enables direct screening of therapeutic antibody gene sequences for serious diseases, such as fully human nanobodies, bifunctional antibodies, and those for CAR-T therapy. The produced fully human nanobody sequences can be used for drug development without further in vitro humanization, thereby saving a lot of time and cost and reducing the risk associated with subsequent drug development. The use of fully human nanobody-producing mice has become an inevitable trend in the development of therapeutic nanobody drugs.
Common techniques for the preparation of animal models with large fragments of genes (more than hundreds of kilobases) being humanized include chromosome engineering, RMCE (recombinase-mediated cassette exchange) and single BAC transgenesis. Chromosome engineering has a high threshold with a development cycle of about 5 years, and relies on embryonic stem cells from the recipient species, limiting its use to species from which highly efficient embryonic stem cells can be isolated, such as mice. The size of the target gene fragment which is recombined into the genome through RMCE technology each time is about 200 kb, and as such, gene modification at the megabase scale requires at least 5 to 6 times of gene recombination on embryonic stem cells, and it will take about 5 years to construct a complete animal model. For the single BAC transgenesis, the transgene size is constrained by the size of BAC. Generally, the gene fragment transferred during a single transgenesis procedure is about 200 kb in size, and it is impossible to transfer genes at megabase scale.
In addition, a fully human nanobody-producing mouse generally exhibits an immune titer lower than a wild mouse, which also implies less diversity in the screened antibody sequences.
Therefore, how to improve the immune titer of a fully human nanobody-producing mouse and how to increase the diversity of antibodies produced therein are technical problems that need to be urgently addressed in the field is.
The information disclosed in the Background Art is only intended to facilitate understanding of the general background of the present disclosure and should not be taken as an acknowledgment or an imply in any form that the information constitutes the prior art that is already known to one of ordinary skill in the art.
In view of the above problems present in the prior art, the present invention aims to provide a method for preparing a genetically modified non-human mammal capable of efficiently producing a humanized antibody, including a whole antibody, a single heavy chain antibody, and a nanobody, a method for preparing a humanized antibody that binds specifically to an antigen by using the non-human mammal produced by the above method, and a method for acquiring a biological sample.
By using the MBGE delivery system, the method for preparing a genetically modified non-human mammal according to the invention can prepare a fully human nanobody-producing mouse with megabase-scale humanized genome fragments within a short period of time (e.g., within six months). Moreover, the efficiency of obtaining positive mice via a single injection is as high as about 15%, which makes it easy to prepare humanized mice with different antibody diversities (e.g., using a variety of strains of mice to increase nanobody sequence diversity). In addition, the prepared non-human mammal has a higher immune titer, allowing for efficient production of a humanized antibody, including a whole antibody, a single heavy chain antibody, and a nanobody.
In order to achieve the purpose of the invention, the technical solutions are provided herein as follows:
In a first aspect, provided herein is a genetically modified non-human mammal, which comprises a disruption in an endogenous heavy chain immunoglobulin gene loci therein.
In some preferred embodiments, the non-human mammal is a mouse, and the disruption in the endogenous heavy chain immunoglobulin gene loci includes deletions of the following gene fragments:
In other preferred embodiments, the non-human mammal is a mouse, and the disruption in the endogenous heavy chain immunoglobulin gene loci includes deletions of the following gene fragments:
The genetically modified non-human mammal as described above can be used as a background animal to prepare a non-human mammal capable of producing a human antibody by introducing appropriate human antibody genes thereinto.
In a second aspect, provided herein is a method for preparing the genetically modified non-human mammal according to the first aspect, which comprises the following steps:
Preferably, the non-human mammal is a mouse.
In some preferred embodiments, the disruption in the endogenous heavy chain immunoglobulin gene loci includes deletions of the following gene fragments:
Further preferably, the deletions of the gene fragments are performed through CRISPR-Cas9 gene editing technique;
In other preferred specific embodiments, the disruption in the endogenous heavy chain immunoglobulin gene loci includes deletions of the following gene fragments:
Further preferably, the deletions of the gene fragments are performed through CRISPR-Cas9 gene editing technique;
In a third aspect, provided herein is a genetically modified non-human mammal, wherein the non-human mammal comprises a disruption in an endogenous heavy chain immunoglobulin gene loci, and the endogenous heavy chain immunoglobulin gene loci comprises a human IGHV gene, a human IGHD gene, a human IGHJ gene as well as an endogenous IgHG2c gene, IgHE gene, IgHA gene, and LCR region of the non-human mammal.
In some preferred embodiments, the non-human mammal is a mouse, and the disruption in the endogenous heavy chain immunoglobulin gene loci includes deletions of the following gene fragments:
In other preferred embodiments, the non-human mammal is a mouse, and the disruption in the endogenous heavy chain immunoglobulin gene loci includes deletions of the following gene fragments:
In feasible embodiments, the human IGHV gene includes at least the following 18 human IGHV genes:
In feasible embodiments, the human IGHD gene includes at least the following 15 human IGHD genes:
In feasible embodiments, the human IGHJ gene includes all the human IGHJ genes; and/or, the endogenous IgHG2c gene of the non-human mammal is the complete endogenous IgHG2c gene, or the endogenous IgHG2c gene fragment without the CH1 domain. When the endogenous IgHG2c gene of the non-human mammal is the complete endogenous IgHG2c gene, after antigen immunization, a humanized whole antibody is produced; when the endogenous IgHG2c gene of the non-human mammal is the endogenous IgHG2c gene fragment without the CH1 domain, after antigen immunization, a humanized single heavy chain antibody is produced.
Preferably, the human IGHV genes, the human IGHD genes, and the human IGHJ genes are operatively linked to each other and subjected to VDJ-rearrangement; further preferably, the operatively linked and/or VDJ-rearranged human IGHV genes, human IGHD genes, and human IGHJ genes, are operatively linked to the endogenous IgHG2c gene, IgHE gene, IgHA gene, and LCR region of the non-human mammal;
Most preferably, the endogenous heavy chain immunoglobulin gene loci of the non-human mammal comprises all the genes shown in Table 13.
In a fourth aspect, provided herein is a method for preparing the genetically modified non-human mammal according to the third aspect, which comprises the following steps:
In feasible embodiments, the non-human mammal is a mouse, and the disruption in the endogenous heavy chain immunoglobulin gene loci as described in step (1) includes deletions of the following gene fragments:
In some other feasible embodiments, the non-human mammal is a mouse, and the disruption in the endogenous heavy chain immunoglobulin gene loci as described in step (1) includes deletions of the following gene fragments:
In some specific embodiments, the non-human mammal is a mouse, and in step (2), the human IGHV gene includes at least the following 18 human IGHV genes:
IGHD1-1, IGHD3-3, IGHD6-6, IGHD1-7, IGHD3-10, IGHD6-13, IGHD1-14, IGHD2-15, IGHD3-16, IGHD5-18, IGHD6-19, IGHD1-20, IGHD3-22, IGHD1-26, and IGHD7-27; optionally, the human IGHD gene further includes one or several or all of the human IGHD genes selected from: IGHD2-2, IGHD2-8, IGHD3-9, IGHD3-10, IGHD4-11, IGHD5-12, IGHD3-16, IGHD4-17, IGHD2-21, IGHD4-23, IGHD5-24, and IGHD6-25; and further optionally, the human IGHD gene further includes one or several or all of the human IGHD genes selected from: IGHD3-3, IGHD3-10, IGHD3-16, and IGHD5-18;
In a preferred embodiment of the above specific embodiments, in step (2), the human IGHV gene, the human IGHD gene, the human IGHJ gene as well as the endogenous IgHG2c gene, IgHE gene, IgHA gene, and LCR region of the non-human mammal are introduced by the following procedure:
Further preferably, in step (2), the human IGHV gene, the human IGHD gene, the human IGHJ gene as well as the endogenous IgHG2c gene, IgHE gene, IgHA gene, and LCR region of the non-human mammal are introduced by the following procedure:
In the non-human mammal prepared according to the above method, the human IGHV genes, the human IGHD genes, and the human IGHJ genes are operatively linked to each other and subjected to VDJ-rearrangement, and the operatively linked and/or VDJ-rearranged human IGHV genes, human IGHD genes, and human IGHJ genes, are operatively linked to the endogenous IgHG2c gene, IgHE gene, IgHA gene, and LCR region of the non-human mammal;
In a fifth aspect, provided herein a method for preparing a humanized whole antibody or a humanized single heavy chain antibody or a humanized nanobody that specifically binds to an antigen, comprising:
In a sixth aspect, provided herein is a method for preparing a humanized single heavy chain antibody or a humanized nanobody that specifically binds to an antigen, comprising:
In a seventh aspect, provided herein is a method for acquiring a biological sample, comprising:
Preferably, the biological sample is selected from spleen tissues, splenocytes, or B cells.
In an eighth aspect, provided herein is a biological sample which is obtained by the method according to the seventh aspect.
In a ninth aspect, provided herein is a sgRNA composition, which comprises: sgRNA for deleting the CH1 fragment of mouse antibody gene heavy chain IgHM; sgRNA for deleting the mouse antibody gene light chain Igκc; and sgRNA for deleting a fragment between IgLc2 and IgLc1 of mouse antibody gene light chain Igλ.
Preferably, the sgRNA for deleting the CH1 fragment of mouse antibody gene heavy chain IgHM comprises a sgRNA as shown in SEQ ID NO: 1 and a sgRNA as shown in SEQ ID NO: 2;
In a tenth aspect, provided herein is a sgRNA composition, which comprises: sgRNA for deleting a fragment between IgHM and IgHA-CH1 of mouse antibody gene heavy chain; sgRNA for deleting the mouse antibody gene light chain Igκc; and sgRNA for deleting a fragment between IgLc2 and IgLc1 of mouse antibody gene light chain Igλ.
Preferably, the sgRNA for deleting the fragment between IgHM and IgHA-CH1 of mouse antibody gene heavy chain comprises a sgRNA as shown in SEQ ID NO: 1 and a sgRNA as shown in SEQ ID NO: 6;
In an eleventh aspect, provided herein is a CRISPR-Cas9 gene editing system, comprising the sgRNA composition according to the ninth or tenth aspect and Cas9 protein.
In a twelfth aspect, provided herein is a gene knockout vector containing DNA sequence(s) encoding the sgRNA(s) in the sgRNA composition according to the ninth or tenth aspect.
In a thirteenth aspect, provided herein is a mouse embryonic stem cell, comprising the gene knockout vector according to the twelfth aspect.
The genetically modified non-human mammals prepared according to the method of the present invention exhibit a higher immune titer, are capable of efficiently producing a humanized single heavy chain antibody or nanobody after antigen immunization, and thereby can serve as an efficient platform for producing a humanized whole antibody or single heavy chain antibody or nanobody. Furthermore, the method for preparing the non-human mammal of the present invention has a relatively high efficiency (about 15%) in obtaining positive animals, thereby making it easy to prepare humanized mice with diverse antibodies, which allows for the realization of nanobody sequence diversity by a variety of strains of mice.
One or more examples are exemplified by the pictures in the accompanying drawings that correspond thereto and are not intended to be limiting of the embodiments. As used herein, the word “exemplary” means “serving as an example, embodiment, or illustrative”. Any embodiment described herein as “exemplary” is not necessarily to be construed as superior or better than other embodiments.
In order to make the purpose, technical solutions and advantages of the embodiments of the invention clearer, the technical solutions in the embodiments of the invention will be described clearly and completely, obviously, the described embodiments are some of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work are within the scope of the present invention.
In addition, in order to better explain the present invention, a lot of specific details are given in the following embodiments. It will be understood by those skilled in the art that the present invention may be practiced without certain specific details. In some embodiments, materials, elements, methods, means, etc., well known to those skilled in the art, are not described in detail so as to highlight the spirit of the present invention.
Throughout the specification and claims, the term “comprising” or variations thereof, such as “including” or “containing”, will be understood to include the stated components and not to exclude other elements or other components, unless expressly indicated otherwise.
In addition, in the following embodiments, the mice used were C57BL/6 mice purchased from Viton Lever, and the starting mice were aged 4 to 6 weeks and weighed about 18 g to 22 g.
To delete the CH1 region of mouse IgHM gene, two target sites were selected, one on the upstream of the first exon of mouse IgHM gene and the other on the downstream thereof, and one sgRNA was designed for each target site.
PCR products were recovered with a recovery kit and used for the subsequent experiment, that is, in vitro transcription.
The above transcription system was incubated in a 37° C. incubator for 18 h and recovered with a kit known as MEGAclear™ Kit Purification for Large Scale Transcription Reactions.
Mice were subjected to ovulation induction and then in vitro fertilization to obtain fertilized eggs, which were cultured; then, sgRNAs and Cas9 protein were mixed together and electroporated into the fertilized eggs of mice, or Cas9 protein (or Cas9 mRNA, commercially available) along with sgRNAs were injected into the fertilized eggs of mice via microinjection.
The resultant fertilized egg cells were implanted into surrogate female mice, thereby producing F0 generation chimeric mice. By extracting genomic DNA from the mouse tail, performing PCR amplification, and analyzing the PCR amplification products, F0 generation positive knockout mice were identified.
In the above PCR amplification, the used PCR primers mIghM-teko-1F and mIghM-teko-1R were designed at both terminals of the deleted sequence. The sequences of the primers were shown in Table 5.
Analyses of PCR amplification products included agarose gel electrophoresis and sequencing analyses so as to determine whether the target sequence had been deleted.
According to the sequencing results of the PCR products, it was determined that the deleted sequence was 323 bp in length. Therefore, the PCR products amplified with the above primers could detect whether the deleted genes or the wild-type genes, depending on the size of the PCR product fragments, wherein for the wild-type genes and those genes with the target sequence deleted, the target bands amplified with the primers mIghM-teko-1F and mIghM-teko-1R were 907 bp and 584 bp in size, respectively (the results were not shown).
F0 generation chimeric mice with correct gene knockout were selected for the subsequent reproduction and identification.
F0 generation mice with target gene knockout were mated with wild-type mice to obtain F1 generation mice. By extracting genomic DNA from the mouse tail and performing PCR detection, stably heritable gene knockout positive F1 generation heterozygous mice were selected. The F1 generation heterozygous mice were then mated with each other to obtain gene knockout positive F2 generation homozygous mice, i.e., IgM knockout homozygous mice. The obtained F2 generation mice and their offspring homozygous mice were subjected to genotype identification in the same way as that in step 1.6 above. Meanwhile, the gene knockout homozygous mouse and heterozygous mouse identified by sequencing were used as a homozygous positive control (the lane labeled “+”) and a heterozygous control (the lane labeled “+/−”), respectively. Furthermore, a wild-type mouse control (the lane labeled “−”) and a negative control without DNA template (also called a “water control”) were also set up. The results were shown in
As shown in
To knock out the mouse Igκc gene, one target on the exon of the mouse Igκc gene was selected, and one sgRNA was designed for this target. The target schematic diagram of sgRNA was shown in
G
PCR products were recovered with a recovery kit and used for the subsequent experiment, that is, in vitro transcription.
The transcription system was incubated in a 37° C. incubator for 18 h and recovered with a kit known as MEGAclear™ Kit Purification for Large Scale Transcription Reactions.
Mice were subjected to ovulation induction and then in vitro fertilization to obtain fertilized eggs, which were cultured; then, the sgRNA and Cas9 protein were mixed together and electroporated into the fertilized eggs of mice, or Cas9 protein (or Cas9 mRNA, commercially available) along with the sgRNA was injected into the fertilized eggs of mice via microinjection.
The resultant fertilized egg cells were implanted into surrogate female mice, thereby producing F0 generation chimeric mice. By extracting genomic DNA from the mouse tail, performing PCR amplification, and analyzing the PCR amplification products, F0 generation positive knockout mice were identified.
In the above PCR amplification, the used PCR primers KC-nF and KC-nR were designed at both terminals of the deleted sequence. The sequences of the primers were shown in Table 7.
Analyses of PCR amplification products included agarose gel electrophoresis and sequencing analyses so as to determine whether the target sequence had been deleted. According to the sequencing results of the PCR products, it was determined that the deleted sequence was 76 bp in length. Therefore, the PCR products amplified with the above primers could detect whether the deleted genes or the wild-type genes, depending on the size of the PCR product fragments, wherein for the wild-type genes and those genes with the target sequence deleted, the target bands amplified with the primers KC-nF and KC-nR were 299 bp and 223 bp in size, respectively (the results were not shown).
F0 generation chimeric mice with correct gene knockout were selected for the subsequent reproduction and identification.
F0 generation mice with target gene knockout were mated with wild-type mice to obtain F1 generation mice. By extracting genome from the mouse tail and performing PCR detection, stably heritable gene knockout positive F1 generation heterozygous mice were selected. The F1 generation heterozygous mice were then mated with each other to obtain gene knockout positive F2 generation homozygous mice, i.e., Igκc knockout homozygous mice. The obtained F2 generation mice and their offspring homozygous mice were subjected to genotype identification in the same way as that in step 2.6 above, and the results thereof were shown in
As shown in
To delete the fragment between IgLc2 and IgLc1 of mouse antibody gene light chain Igλ, one target on the upstream of the exon of mouse IgLc2 gene and one target on the downstream of the exon of mouse IgLc1 gene were selected, respectively, and one sgRNA was designed for each target.
3. Construction of IgL mice
PCR products were recovered with a recovery kit and used for the subsequent experiment, that is, in vitro transcription.
The transcription system was incubated in a 37° C. incubator for 18 h and recovered with a kit known as MEGAclear™ Kit Purification for Large Scale Transcription Reactions.
Mice were subjected to ovulation induction and then in vitro fertilization to obtain fertilized eggs, which were cultured; then, the sgRNAs and Cas9 protein were mixed together and electroporated into the fertilized eggs of mice, or Cas9 protein (or Cas9 mRNA, commercially available) along with the sgRNAs were injected into the fertilized eggs of mice via microinjection.
The resultant fertilized egg cells were implanted into surrogate female mice, thereby producing F0 generation chimeric mice. By extracting genomic DNA from the mouse tail, performing PCR amplification, and analyzing the PCR amplification products, the F0 generation positive knockout mice were identified.
In the above PCR amplification, the used PCR primers LC2-nF1 and IgL-R2 were designed at both terminals of the deleted sequence. The sequences of the primers were shown in Table 9.
Analyses of PCR amplification products included agarose gel electrophoresis and sequencing analyses so as to determine whether the target sequence had been deleted. According to the sequencing results of the PCR products, it was determined that the deleted sequence was 137 kp in length. Therefore, the PCR product size of the knockout mice amplified with the above primers was 1170 bp. The PCR primer LC1-nF21 was designed near the 3′ end of the deleted sequence, and the primers LC1-nF21 and IgL-R2 were used to detect the target fragment without gene deletion, and the PCR product size was 1424 bp. Depending on the size of the PCR product fragment, it is also possible to determine whether it is a wild-type gene or a gene with the target sequence deleted.
F0 generation chimeric mice with correct gene knockout were selected for the subsequent reproduction and identification.
F0 generation mice with target gene knockout were mated with wild-type mice to obtain F1 generation mice. By extracting genome from the mouse tail and performing PCR detection, stably heritable gene knockout positive F1 generation heterozygous mice were selected. The F1 generation heterozygous mice were then mated with each other to obtain gene knockout positive F2 generation homozygous mice, i.e., IgL knockout homozygous mice. The obtained F2 generation mice and their offspring homozygous mice were subjected to genotype identification in the same way as that in step 3.6 above, and the results thereof were shown in
As shown in
To delete the fragment between IgHM and IgHA-CH1 of mouse antibody gene heavy chain, one target on the upstream of the exon of mouse IgHM gene and one target on the downstream of the first exon of mouse IgHA gene were selected, respectively, and one sgRNA was designed for each target.
PCR products were recovered with a recovery kit and used for the subsequent experiment, that is, in vitro transcription.
The transcription system was incubated in a 37° C. incubator for 18 h and recovered with a kit known as MEGAclear™ Kit Purification for Large Scale Transcription Reactions.
Mice were subjected to ovulation induction and then in vitro fertilization to obtain fertilized eggs, which were cultured; then, sgRNAs and Cas9 protein were mixed together and electroporated into the fertilized eggs of mice, or Cas9 protein (or Cas9 mRNA, commercially available) along with the sgRNAs were injected into the fertilized eggs of mice via microinjection.
The resultant fertilized egg cells were implanted into surrogate female mice, thereby producing F0 generation chimeric mice. By extracting genomic DNA from the mouse tail, performing PCR amplification, and analyzing the PCR amplification products by gel electrophoresis and sequencing, F0 generation positive knockout mice were identified.
In the above PCR amplification, the used PCR primers IgA-KO-1F and IgA-KO/WT-1R were designed at both terminals of the deleted sequence. The sequences of the primers were shown in Table 11.
The PCR amplification products were analyzed by agarose gel electrophoresis and sequencing, respectively, to determine whether the target sequence had been deleted.
According to the sequencing results of the PCR products, it was determined that the deleted sequence was 163 kb in length. The PCR product size of the gene knockout mice amplified with the above primers was 1170 bp. The PCR primer IgA-KO/WT-1R was designed near the 3′ end of the deleted sequence, and the primers IgA-WT-1F and IgA-KO/WT-1R were used to detect the target fragment without gene deletion, and the PCR product size was 724 bp. Depending on the size of the PCR product fragment, it is also possible to determine whether it is a wild-type gene or a gene with the target sequence deleted.
F0 generation chimeric mice with correct gene knockout were selected for the subsequent reproduction and identification.
F0 generation mice with target gene knockout were mated with wild-type mice to obtain F1 generation mice. By extracting genome from the mouse tail and performing PCR detection, stably heritable gene knockout positive F1 generation heterozygous mice were selected. The F1 generation heterozygous mice were then mated with each other to obtain gene knockout positive F2 generation homozygous mice, i.e., IgA knockout homozygous mice. The obtained F2 generation mice and their offspring homozygous mice were subjected to genotype identification in the same way as that in step 4.6 above. Meanwhile, the gene knockout homozygous mouse identified by sequencing were used as a homozygous positive control (the lane labeled “+”). Furthermore, a wild-type mouse control (the lane labeled “−”) and a negative control without DNA template (also called a “water control”) were also set up, and the results thereof were shown in
As shown in
The IgM knockout homozygous mice prepared in Example 1 were mated with the IgK knockout homozygous mice prepared in Example 2 to obtain IgM-IgK knockout heterozygous mice; simultaneously, the IgM knockout homozygous mice prepared in Example 1 were mated with the IgL knockout homozygous mice prepared in Example 3 to obtain IgM-IgL knockout heterozygous mice; then the obtained IgM-IgK knockout heterozygous mice were mated with the IgM-IgL knockout heterozygous mice to obtain IgM(+/+)IgK(+/−)IgL(+/−) mice; finally, the IgM(+/+)IgK(+/−)IgL(+/−) mice were mated with each other to obtain IgM(+/+)IgK(+/+)IgL(+/+) mice, called IgM-KL mice.
The target mouse strain was identified by extracting genomic DNA from the mouse tail, performing PCR amplification, and analyzing the PCR amplification products by gel electrophoresis. The primers for identifying IgM, IgK, and IgL gene deletions were the same as those disclosed in Examples 1, 2, and 3, respectively. Meanwhile, the gene knockout homozygous mouse and heterozygous mouse with the corresponding gene knockout identified by sequencing were used as a homozygous positive control (the lane labeled “+”) and a heterozygous control (the lane labeled “+/−”), respectively. Furthermore, a wild-type mouse control (the lane labeled “−”) and a negative control without DNA template (also called a “water control”) were also set up, and the genotype identification results were shown in
As shown in
The IgM-KL knockout homozygous mice prepared in Example 5 were mated with the IgA knockout homozygous mice prepared in Example 4 to obtain IgA-KL knockout heterozygous mice; then the obtained IgA-KL knockout heterozygous mice were mated with each other to obtain IgA (+/+) IgK(+/+)IgL(+/+) mice, called IgA-KL mice.
The target mouse strain was identified by extracting genomic DNA from the mouse tail, performing PCR amplification, and analyzing the PCR amplification products by gel electrophoresis. The primers for identifying IgA and IgK deletions were the same as those disclosed in Examples 4 and 2, respectively, and the primers for identifying IgL deletion were shown in Table 12 below. Meanwhile, the gene knockout homozygous mouse with the corresponding gene knockout identified by sequencing was used as a homozygous positive control (the lane labeled “+”). Furthermore, a wild-type mouse control (the lane labeled “−”) and a negative control without DNA template (also called a “water control”) were also set up, and the genotype identification results were shown in
As shown in
In this example, 6 BACs carrying all human or murine antibody gene sequences to be introduced were introduced into the IgM-KL mice prepared in Example 5, to prepare IgM-KL-hIgHD mice and IgA-KL-hIgHD mice. The six BACs were called CH17-268I9, CTD-3054M17, CTD-2548B8-CZ, RP11-965B13, CH17-185P21-CZ, and RP23-351J19-CZ, respectively, among which, the four BACs CH17-268I9, CTD-3054M17, CTD-2548B8-CZ, and RP11-965B13 carried a total of about 70 V genes, CH17-185P21-CZ contained the complete human D region genes and J region genes, and RP23-351J19-CZ contained murine IgHG2c (a complete IgHG2c region for producing conventional antibodies; a IgHG2c fragment without CH1 for producing nanobodies; in this example, the IgHG2c fragment without CH1 was introduced), IgHE, IgHA, and LCR region (35 kb) (for detailed information of the genes carried by each BAC, please see Table 13); the two parts above were connected to each other via a Switch region of the murine IgHM (i.e., between human J region and murine IgHG2c genes).
Of the six BAC clones above, CH17-268I9, CTD-3054M17, and RP11-965B13 were the original BAC clones purchased from INVITROGEN (Shanghai) Trading Co. Ltd., and while CTD-2548B8-CZ, RCH17-185P21-CZ, and RP23-351J19-CZ were obtained by making some modifications on the basis of their respective original BAC clone strains (i.e., CTD-2548B8, RCH17-185P21, and RP23-351J19, also purchased from INVITROGEN (Shanghai) Trading Co. Ltd.), and the specific modification method was as follows.
Firstly, the blank BAC strain was prepared into electrocompetent cells. Then, pKD46-Tet plasmids (Wuhan Miaoling Bio Co., Ltd., P7957) were electroporated into the electrocompetent cells. The fragments to be recombined (as shown in SEQ ID NO:76-78, respectively) were constructed by OverLap-PCR or enzymatic ligation. The BAC bacteria carrying pKD46-Tet plasmids were prepared into electrocompetent cells, and then the fragments to be recombined were electrotransferred into the competent cells. Subsequently, screening was carried out by using the corresponding antibiotics, and the positive clones were verified by colony PCR. Finally, the positive BAC clones were extracted from the bacteria and determined by Fast-NGS sequencing.
In Table 13, “mIgHG2c-CH1” represents mIgHG2c gene sequence without the CH1 fragment.
The specific procedure was as follows:
(1) 6 BACs carrying all the human antibody gene sequences to be introduced were introduced into the IgM-KL mice prepared in Example 5; specifically, BAC plasmids were extracted firstly, and then the BAC framework was cut off with the corresponding restriction endonuclease followed by purification; the purified BAC genes were then mixed in equal molar amounts to prepare an injection solution, which was then injected into the masculonucleus of fertilized eggs through the pronucleus, and finally the fertilized eggs were transplanted into the surrogate mice to obtain F0 generation mice with human antibody genes introduced. Subsequently, the introduced antibody gene sequences were identified by PCR. The primers used were shown in Table 14.
(2) the F0 generation mice with human antibody gene sequences introduced obtained in step (1) were mated with IgM-KL homozygous mice to obtain F1 generation heterozygous mice. By extracting genomic DNA from the mouse tail and performing PCR detection, stably heritable gene recombined positive F1 generation heterozygous mice were selected. The F1 generation heterozygous mice were then mated with each other to obtain gene recombined positive F2 generation homozygous mice, i.e., IgM-KL-hIgHD homozygous mice. The obtained F2 generation homozygous mice were subjected to genotype identification in the same way as that in step 1 above. At the same time, the IgM-KL background mice were detected by PCR, and the identification method was the same as that in Example 5.
The results were shown in
The above results showed that all the above genes were successfully introduced, i.e., IgM-KL-hIgHD mice were successfully obtained in this example.
In this example, the IgM-KL-hIgHD mice prepared in Example 7 were mated with the IgA-KL homozygous mice to obtain F1 generation mice, i.e., IgA-KL-hIgHD heterozygous mice; then the F1 generation heterozygous mice were mated with each other to obtain F2 generation homozygous mice, i.e., IgA-KL-hIgHD mice.
The results of genotype identification of the F2 generation IgA-KL-hIgHD mice were shown in
The above results showed that all the above genes were successfully introduced, i.e., IgA-KL-hIgHD mice were successfully obtained in this example.
Three IgM knockout homozygous mice (prepared in Example 1) were individually immunized with OVA (chicken ovalbumin, purchased from Beijing Borsee Science and Technology Co., Ltd.) as an antigen, and the procedure was specifically as follows:
Mice aged 6-8 weeks were selected. The antigen was mixed with an equal volume of Freund's complete adjuvant and emulsified until it no longer dissolved in water. Then the emulsion can be injected subcutaneously at multiple sites into the mice. For the primary immunization, the injection dose was 50 μg per mouse and the injection volume was 0.2 mL per mouse.
Two weeks after the primary immunization, a second immunization was carried out subcutaneously. The antigen was emulsified with an equal volume of Freund's incomplete adjuvant and then injected subcutaneously at multiple sites into the mice. The injection dose was reduced to 25 μg per mouse and the injection volume was 0.2 mL per mouse.
Blood samples were collected on days 0, 17, and 24, respectively, a plate was coated with goat anti-mouse IgM polyclonal antibodies, the antigen was labeled with biotin, and the IgM antibody titer in serum was detected using HRP-Streptavidin. The antigen immune titer assay results of the three IgM knockout homozygous mice were shown in
As shown in
(II) Western Blot Assay of Serum from IgM Gene Knockout Mice Immunized with CRP
CRP (i.e., human C-reactive protein, purchased from Beijing Biobridge Biotechnology Co., Ltd.) antigen affinity column material was prepared according to the instructions of CNBr-activated Sepharose™ 4B (purchased from GE, No. 17043001), and the volume of CRP-Sepharose packing prepared using 1.5 mg of CRP antigen was set to 1.5 mL.
One C57BL/6 wild-type mouse (purchased from Viton Lever), one IgM knockout heterozygous mouse (the F1 generation heterozygous mouse prepared in Example 1), and one IgM knockout homozygous mouse (the F2 generation homozygous mouse prepared in Example 1), each aged 6-8 weeks, were immunized with CRP, and the immunization process was the same as that with OVA immunization. On the day 7 after the third immunization, 20-50 μL of mouse blood was collected, and placed at room temperature for about 30 min until the blood was coagulated, and the serum was collected after centrifugation. 1 μL of each sample was taken separately, to which 100 μL of PBS was added, followed by addition of 10 μL of CRP-Sepharose packing. After reacting at room temperature for 60 min, centrifugation was carried out at 6000 rpm for 30 seconds, and then the supernatant was discarded. The packing was washed with PBS for 3 times, resuspended with 10 μL PBS and boiled, subjected to 12% SDS-PAGE electrophoresis, and transferred onto a PVDF membrane and blocked, then reacted with goat anti-mouse IgM antibody (Sigma, ISO2-1KT) and further color developed, and the results were shown in
As shown in
(III) RT-PCR Detection of IgM Gene Knockout Mice Immunized with CRP
Splenocytes were taken from the IgM knockout homozygous mice after the third immunization with CRP antigen in the step (II) above, and total RNA was extracted with Trizol and subjected to reverse transcription to cDNA. With the cDNA as a template, the variable region and part of the CH2 gene linked thereto were amplified using primers specific for IgM subtype antibody, with the upstream primer being MHV1-12 mixed primers and the downstream primer being IgHM-CH2-R4. The primers used and their sequences were shown in Table 15, wherein the variable bases S, Y, R, W, M, and K were as defined in the prior art; specifically, S was G or C, Y was C or T, R was A or G, W was A or T, M was A or C, and K was G or T.
The PCR reaction system was shown in Table 16.
The PCR reaction procedure was shown in Table 17.
The PCR amplification products were ligated into a pEASY®-Blunt Zero Cloning vector, and then transformed into TOP10 strain and plated (ampicillin resistant). One clone was picked for colony PCR, and the results were shown in
IgA knockout homozygous mice (prepared from Example 4) were immunized with CRP (human C-reactive protein) and OVA (chicken ovalbumin) as an antigen. The immune method was as follows:
Mice aged 6-8 weeks were selected. The antigen was mixed with an equal volume of Freund's complete adjuvant and emulsified until it no longer dissolved in water. Then the emulsion can be injected subcutaneously at multiple sites into the mice. For the primary immunization, the injection dose was 50 μg per mouse and the injection volume was 0.2 mL per mouse. After the primary immunization, subsequent subcutaneous immunizations were carried out every two weeks. The antigen protein was emulsified with an equal volume of Freund's incomplete adjuvant and then injected subcutaneously at multiple sites into the mice. The injection dose was reduced to 25 μg per mouse and the injection volume was 0.2 mL per mouse.
The serum titer detection method was performed as follows: the antigen protein was diluted to 2 μg/mL, 100 μL of the dilution was taken and added to a polystyrene enzyme-linked plate for plating, and the IgA antibody that specifically binds to the antigen protein in serum was detected using Biotin-goat anti-mouse IgA (Abcam, ab97231).
The results were shown in
(II) Western Blot Assay of Serum from IgA Knockout Homozygous Mice Immunized with CRP
CRP antigen affinity column material was prepared according to the instructions of CNBr-activated Sepharose™ 4B (purchased from GE, No. 17043001), and the volume of CRP-Sepharose packing prepared using 1.5 mg of CRP antigens was set to 1.5 mL.
Two C57BL/6 wild-type mice and one IgA knockout homozygous mouse (prepared from Example 4), each aged 6-8 weeks, were immunized with CRP, i.e. human C-reactive protein, and the immunization procedure was the same as that in the above step (I). On the day 7 after the third immunization, 20-50 μL of mouse blood was collected, and placed at room temperature for about 30 min until the blood was coagulated, and the serum was collected after centrifugation. 2 μL of each sample was taken separately, to which 100 μL of PBS was added, followed by addition of 10 μL of CRP-Sepharose packing. After reacting at room temperature for 60 min, centrifugation was carried out at 6000 rpm for 30 seconds, and then the supernatant was discarded. The packing was washed with PBS for 3 times, resuspended with 10 μL PBS and boiled, subjected to 12% SDS-PAGE electrophoresis, and transferred onto a PVDF membrane and blocked, then reacted with goat anti mouse IgG Fc HRP (JACKSON,115-035-071) and further color developed.
According to the design of the present application, since the mouse genes IgM, IgD, IgG, and IgE were knocked out and the CH1 gene of IgA heavy chain was knocked out, immunization of IgA mice with an antigen protein will only produce IgA heavy chain antibodies (without the CH1 domain), and the molecular weight of one single heavy chain of IgA was about 40 KD. Antibodies produced by C57BL/6 wild-type mice and IgA homozygous mice after immunization with the antigen protein were separated, electrophoresed, and color developed, and the results were shown in
(III) RT-PCR Detection of Splenocytes from IgA Homozygous Mice Immunized with CRP
Splenocytes were taken from the IgA homozygous mice after the third immunization with CRP antigen as described in the step (II) above, total RNA was extracted with Trizol and subjected to reverse transcription to cDNA. With the cDNA as a template, the variable region and part of the CH2 gene linked thereto were amplified using primers specific for IgA subtype antibody, with the upstream primer being MHV1-12 mixed primers (the sequences thereof were shown in table 15 above) and the downstream primer being IgHA-CH2-R3 (the sequence was: CTGCATCCTTCCCAGTGGAG, i.e., SEQ ID NO: 61)
The PCR reaction system was the same as that shown in Table 16 above (except for the different downstream primer).
The PCR reaction procedure was the same as that shown in Table 17 above.
The PCR amplification products were ligated into a pEASY®-Blunt Zero Cloning vector, and then transformed into TOP10 strain and plated (ampicillin resistant). Four clones were picked for colony PCR, and the results were shown in
Wild-type C57BL/6, IgM-KL homozygous mice (prepared from Example 5), IgA homozygous mice (prepared from Example 4, as a control), and IgA-KL homozygous knockout mice (prepared from Example 6) were immunized with OVA (chicken ovalbumin), respectively, with the specific procedure as follows:
Mice aged 6-8 weeks were selected. The antigen was mixed with an equal volume of Freund's complete adjuvant and emulsified until it no longer dissolved in water. Then the emulsion can be injected subcutaneously at multiple sites into the mice. For the primary immunization, the injection dose was 50 μg per mouse and the injection volume was 0.2 mL per mouse.
Two weeks after the initial immunization, a second immunization was carried out subcutaneously. The antigen was emulsified with an equal volume of Freund's incomplete adjuvant and then injected subcutaneously at multiple sites into the mice. The injection dose was reduced to 25 μg per mouse and the injection volume was 0.2 mL per mouse.
The serum titer detection was performed as follows: the antigen protein was diluted to 2 μg/mL, and 100 μL of the dilution was taken and added to a polystyrene enzyme-linked plate for plating. For the IgM-KL mice, goat anti-mouse IgM antibody (Sigma, ISO2-1KT) was used, and for the IgA-KL mice, Biotin-goat anti-mouse IgA (Abcam, ab97231) was used, to detect the IgA antibody that specifically binds to the antigen protein in serum.
The results were shown in
(II) Western Blot Assay of Serum from IgM-KL & IgA-KL Homozygous Mice Immunized with OVA
Sera from four kinds of mice, IgM, IgM-KL, IgA, and IgA-KL, after three immunizations using OVA were taken and subjected to Coomassie Brilliant Blue staining to examine their protein abundance. The results were shown in
The loading volume of the serum samples in each group of tests was 0.5 μL. After boiling for denaturation, the samples were loaded onto a 12% SDS-PAGE gel for electrophoresis, and transferred onto a PVDF membrane and blocked, the goat anti-mouse Kappa polyclonal antibody (HRP*Polyclonal Goat Anti-Mouse Kappa, C030214) and the goat anti-mouse Lamda polyclonal antibody (HRPPolyclonal Goat Anti-Mouse Lamda, C030213) from Leibochem (Shanghai) Biochemical Technology Co., Ltd. were used to detect the expression of light chain proteins in the mouse sera as described above. The results were shown in
As can be seen in
IgM-KL-hIgHD homozygous mice were immunized with GCC-mFc protein. Specifically, mice aged 6-8 weeks were selected. The antigen was mixed with an equal volume of Freund's complete adjuvant and emulsified until it no longer dissolved in water. Then the emulsion can be injected subcutaneously at multiple sites into the mice. For the primary immunization, the injection dose was 100 μg per mouse and the injection volume was 200 μL per mouse.
Two weeks after the primary immunization, a second immunization was carried out subcutaneously. The antigen was emulsified with an equal volume of Freund's incomplete adjuvant and then injected subcutaneously at multiple sites into the mice. The injection dose was reduced to 50 μg per mouse and the injection volume was 200 μL per mouse. The specific immunization scheme was shown in Table 18.
The serum titer detection was performed as follows: the antigen protein was diluted to 2 μg/mL, 100 μL of the dilution was taken and added to a polystyrene enzyme-linked plate for plating, and the humanized nanobody that specifically binds to the antigen protein in the serum from IgM-KL-hIgHD mice was detected using goat anti mouse IgG Fc HRP (JACKSON, 115-035-071) antibody.
The results were shown in
(II) Western Blot Assay of Serum from the IgM-KL-hIgHD Homozygous Mice Immunized with GCC-mFc
Sera from IgM-KL-hIgHD mice (#3492, #3493, #3304) after the fifth immunization using GCC-mFc and unimmunized C57BL/6 mice (#3347) were taken for Western assay to verify whether human-mouse chimeric single heavy chain antibody was expressed in the IgM-KL-hIgHD homozygous mice.
The loading volume of the serum samples in each group of tests was 0.5 μL. After boiling for denaturation, the samples were loaded onto a 12% SDS-PAGE gel for electrophoresis, and transferred onto a PVDF membrane and blocked, and goat anti-mouse IgG Fc HRP (JACKSON, 115-035-071) was used to detect the expression of human-mouse chimeric nanobodies in the mouse serum as described above. The results were shown in
As shown in
One unimmunized mouse and one GCC-mFc-immunized IgM-KL-hIgHD homozygous mouse were selected, and splenocytes from the mice were taken for RNA extraction, and then RNA was reverse transcribed into cDNA using a reverse transcription kit (Takara, 6110A). PCR amplification was performed by using the combination of 10 primers for human V genes with three specific primers for human J genes, respectively, with the cDNA of the sample as a template, to obtain the heavy chain variable region fragments which were then subjected to subsequent sequencing. The sequences of the human gene specific primers were shown in Table 19.
Since the PCR products of the two samples required NGS sequencing and data analysis, specific barcode sequences needed to be added at the 5′ end of primers in Table 16. Therefore, AGTGCT was added to the 5′ end of the primer F, and AGACTG was added to the 5′end of the primer R used for the IgM-KL-hIgHD unimmunized mice; GTCTAA was added to the 5′end of the primer F and ATTACT was added to the 5′ end of the primer R used for the IgM KL-hIgHD immunized mice. The samples of IgM-KL-hIgHD were amplified by PCR using primers added with barcode sequences, and the results were shown in
Next, the PCR products were subjected to NGS sequencing.
The sequencing results and human immunoglobulin sequences were analyzed by bioinformatics technology to identify the expression of human VH, DH, JH genes after V(D)J recombination. As a result, of the 1323424 valid sequencing Reads from the unimmunized IgM-KL-hIgHD mouse samples, the expression of most VH gene fragments, all DH genes, and all JH gene fragments were detected (Table 20). Among these gene fragments, some VH genes were located near the constant region, while others were farther from it. The data of Table 20 indicated that human VH, DH, and JH genes on the human-mouse chimeric nanobody genes transferred by the hIgHD scheme can be rearranged and expressed in IgM-KL background mice.
Likewise, of the 1239828 valid sequencing Reads from the immunized IgM-KL-hIgHD mouse samples, the expression of most VH gene fragments, all DH genes, and all JH gene fragments were detected (Table 21).
Comparison of Table 20 with Table 21 showed that the proportions of some VH, DH, and J H genes were significantly up-regulated or decreased before and after the immunization, indicating that the humanized nanobody-producing mouse had a good response to antigen immunity.
IgA-KL-hIgHD homozygous mice and IgA-KL mice were immunized with the CD93 protein. Specifically, mice aged 6-8 weeks were selected. The antigen was mixed with an equal volume of Freund's complete adjuvant and emulsified until it no longer dissolved in water. Then the emulsion can be injected subcutaneously at multiple sites into the mice. For the primary immunization, the injection dose was 100 μg per mouse and the injection volume was 200 μL per mouse.
Two weeks after the primary immunization, a second immunization was carried out subcutaneously. The antigen was emulsified with an equal volume of Freund's incomplete adjuvant and then injected subcutaneously at multiple sites into the mice. The injection dose was reduced to 50 μg per mouse and the injection volume was 200 μL per mouse. The specific immunization scheme was shown in Table 22.
The serum titer detection was performed as follows: the antigen protein was diluted to 2 μg/mL, 100 μL of the dilution was taken and added to a polystyrene enzyme-linked plate for plating, and the humanized nanobody that specifically binds to the antigen protein in sera of IgA-KL-hIgHD mice and IgA-KL mice was detected using goat anti-mouse IgG Fc HRP (JACKSON, 115-035-071) antibody.
As shown in
(II) Western Blot Assay of Serum from IgA-KL-hIgHD Homozygous Mice Immunized with CD93
Sera from IgA-KL-hIgHD mice (#3756, #3577), IgA-KL(#3702, #3706) after the fifth immunization with CD93 and unimmunized C57BL/6 mice (#10252) were taken for Western assay to verify whether human-mouse chimeric nanobodies were expressed in IgA-KL-hIgHD homozygous mice.
The loading volume of the serum samples in each group of tests was 0.2 μL. After boiling for denaturation, the samples were loaded onto a 12% SDS-PAGE gel for electrophoresis, and then transferred onto a PVDF membrane and blocked, goat anti-mouse IgG Fc HRP (JACKSON, 115-035-071) was used to detect the expression of human-mouse chimeric nanobodies in the mouse serum as described above. The results were shown in Table 29 below.
As seen from
One unimmunized mouse and one CD93-immunized IgA-KL-hIgHD homozygous mouse were selected, and splenocytes from the mice were taken for extracting RNA, and the RNA was then reverse transcribed into cDNA using a reverse transcription kit (Takara, 6110A). PCR amplification was performed by using the combination of 10 primers for human V genes with three specific primers for human J genes, respectively, with the cDNA of the sample as a template, to obtain the heavy chain variable region fragments which were then subjected to subsequent sequencing. The sequences of the human gene specific primers were shown in Table 19 above.
Since the PCR products of the two samples required NGS sequencing and data analysis, specific barcode sequences needed to be added at the 5′ end of primers in Table 19. Therefore, ACAGAG was added to the 5′ end of the primer F, and AGACTG was added to the 5′end of the primer R used for the IgA-KL-hIgHD unimmunized mice; AGTGCT was added to the 5′end of the primer F and TCCGGA was added to the 5′ end of the primer R used for the IgA KL-hIgHD immunized mice. The samples of IgA-KL-hIgHD were amplified by PCR using primers added with barcode sequences, and the PCR amplification products were subjected to agarose gel electrophoresis. The results were shown in
Next, the PCR products were subjected to NGS sequencing.
The sequencing results and human immunoglobulin sequences were analyzed by bioinformatics technology to identify the expression of human VH, DH, JH genes after V(D)J recombination. As a result, of the 895880 valid sequencing Reads from the unimmunized IgA-KL-hIgHD mouse samples, the expression of most VH gene fragments, all DH genes, and all JH gene fragments were detected (Table 23). Among these gene fragments, some VH genes were located near the constant region, while others were farther from it. The data of Table 23 indicated that human VH, DH, and JH genes on the human-mouse chimeric nanobody genes transferred by the hIgHD scheme can be rearranged and expressed in IgM-KL background mice.
Likewise, of the 877915 valid sequencing Reads from the immunized IgA-KL-hIgHD mouse samples, the expression of most VH gene fragments, all DH genes, and all JH gene fragments was detected (Table 24).
Comparison of Table 23 with Table 24 showed that the proportions of some VH, DH, and J H genes were significantly up-regulated or decreased before and after the immunization, indicating that the humanized nanobody-producing mouse had a good response to antigen immunity.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by one of ordinary skill in the art that the technical solutions described in the foregoing embodiments can still be modified or some technical features can be equivalently substituted; however, these modifications or substitutions do not make the essence of the corresponding technical solutions departing from the spirit and scope of the technical solutions of various embodiments of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202410082783.6 | Jan 2024 | CN | national |
