WO2017113181A1 - Igg hybrid bispecific antibody against tnfα and il-17a - Google Patents

Abstract

The present invention provides an IgG hybrid bispecific antibody against TNFα and IL-17A. The hybrid antibody comprises a first heavy chain and a first light chain from a human IgG binding specifically to TNFα, and a second heavy chain and a second light chain of a human IgG binding specifically to IL-17A. A variable region of the second heavy chain is exchanged with a variable region of the second light chain, and/or a variable region of the first heavy chain is exchanged with a variable region of the first light chain; and constant regions of the first heavy chain and the second heavy chain interact with each other via enhanced electrostatic interaction or hydrophobic interaction.

Description

IgG hybrid anti-TNFα and IL-17A bispecific antibodies Technical field

The present invention relates to the field of immunology, and in particular to IgG hybrid anti-TNFα and IL-17A bispecific antibodies capable of simultaneously binding tumor necrosis factor alpha (TNFα) and interleukin 17A (IL-17A).

Background technique

Monoclonal antibodies produced by genetic engineering currently play an important role in the biomedical field, but many major diseases are not caused by a single antigen, such as tumors and autoimmune diseases, often caused by a variety of cytokines ( Including tumor necrosis factor alpha (TNFα) and interleukin 17A (IL-17A), monoclonal antibodies have certain limitations for the treatment of such diseases. Therefore, if they can be produced, two or more types can be combined. Antigen antibodies can greatly reduce the limitations of monoclonal antibodies to treat diseases, and are very beneficial to the treatment of tumors and autoimmune diseases.

Bispecific antibodies contain two different antigen binding sites and are antibodies that can bind two different antigen molecules. Bispecific antibodies have developed rapidly in the biomedical field and have achieved great success in biomedical fields, such as Treatment of tumors and autoimmune diseases. In the clinical application of bispecific antibodies, many require a complete IgG structure, so the construction of the bispecific antibody vector and the expression of the host cell play a decisive role in its efficacy and clinical application.

Currently, host systems widely used to study antibody expression are E. coli and mammalian cells. However, many of the reported antibody expression systems such as Escherichia coli, yeast, and insect cells have disadvantages such as low protein activity, inability to perform continuous expression, and strict requirements on the purification process. Mammalian cells have the ability to efficiently express endogenous heavy and light chain genes, glycosylate, correctly fold and assemble antibodies, and secrete active antibodies, facilitating the identification of antibody affinity, specificity, and the like. The HEK293F expression system has accurate post-transcriptional modification function, and the expressed protein is close to the natural protein molecule; it has the extracellular secretion function of the product, and rarely secretes its own endogenous protein, which facilitates the separation and purification of downstream products; High-density cultures were achieved in serum-free medium with higher yields.

Although the configuration and variety of bispecific antibodies are increasing, there are many corresponding problems with existing bispecific antibodies. In terms of configuration, many configurations of existing bispecific antibodies differ greatly from naturally occurring antibody configurations, resulting in poor stability, poor specificity, easy generation of homodimers, and no good pharmacokinetics. Features, even produce immunogenicity. Therefore, it is one of the technical problems to be solved in the art to provide an IgG hybrid type anti-TNFα and IL-17A bispecific antibody which is close to the molecular weight and configuration of IgG of human body.

Summary of the invention

One of the objects of the present invention is to provide an IgG hybrid anti-TNFα and IL-17A bispecific antibody or a biologically active fragment derived from the antibody capable of specifically binding TNFα and IL-17A.

Another object of the present invention is to provide a nucleotide sequence encoding the IgG hybrid type anti-TNFα and IL-17A bispecific antibodies.

Another object of the present invention is to provide a mammalian cell line which secretes the IgG hybrid type anti-TNFα and IL-17A bispecific antibodies of the present invention.

Another object of the present invention is to provide a method of producing the IgG hybrid anti-TNFα and IL-17A bispecific antibodies of the present invention.

Another object of the present invention is to provide a pharmaceutical composition comprising the IgG hybrid type anti-TNFα and IL-17A bispecific of the present invention or a biologically active fragment thereof.

Another object of the present invention is to provide the use of the IgG hybrid anti-TNFα and IL-17A bispecific antibodies of the present invention or biologically active fragments thereof or the pharmaceutical compositions thereof.

Another object of the present invention is to provide a kit for detecting the level of TNFα or IL-17A.

In order to achieve the above object, in one aspect, the present invention provides an IgG hybrid anti-TNFα and IL-17A bispecific antibody or a biologically active fragment derived from the bispecific antibody capable of specifically binding TNFα and IL-17A, The bispecific antibody has a human IgG type for specifically binding to the first heavy chain and the first light chain of TNFα, and a human IgG type for the second heavy chain and the second light for specifically binding IL-17A chain;

Wherein the variable region in the first heavy chain is a variable region of a light chain in an antibody of human IgG-type TNFα; the variable region in the first light chain is an antibody to the human IgG-type TNFα Variable region in the heavy chain; and/or

The variable region in the second heavy chain is a variable region in the light chain of a human IgG type IL-17A antibody; the variable region in the second light chain is an antibody to the human IgG type IL-17A Variable region in the heavy chain;

The constant regions in the first heavy chain and the second heavy chain are bonded to each other by enhanced electrostatic interaction and/or hydrophobic interaction.

Preferably, the enhanced electrostatic effect is by performing a D360K and/or D403K mutation on the constant region in the heavy chain of the antibody of human IgG-type TNFα, and the heavy chain of the antibody of human IgG-type IL-17A is The constant region is achieved by K402D and/or K419D mutations.

More preferably, the heavy chain of the human IgG type TNFα antibody is SEQ ID NO: 1, and the amino acids 360 and 403 in the heavy chain of the human IgG type TNFα antibody are replaced by Asp to Lys; The heavy chain of the antibody of human IgG type IL-17A is SEQ ID NO: 2, and the positions 402, 419 in the heavy chain of the antibody of human IgG type IL-17A are replaced by Lys to Asp.

Since the natural antibody is a homodimer, in order to generate a heterodimer and the expression level of the heterodimer is much larger than that of the homodimer, the present invention solves the heterologous two of the heavy chain. Technical problem of binding of a polymer and binding of a light chain heterodimer, in particular, the present invention allows the binding of the heterologous first heavy chain to the second heavy chain to be greater by enhanced electrostatic or hydrophobic interaction Binding between homologous heavy chains, for example, by site-directed mutagenesis of the Fc fragment of an antibody, alters the Fc-segment of the antibody, allowing homodimers to repel each other, and heterodimer interaction is enhanced; Specifically, for example, the Fc segment of the heavy chain of the antibody of human IgG-type TNFα is subjected to mutation of D360K/D403K, and the Fc segment of the heavy chain of the antibody of human IgG-type IL-17A is subjected to mutation of K402D/K419D, thereby The first heavy chain and the second heavy chain are combined by electrostatic interaction to form a heterodimer. In order to prevent a mismatch between the light and heavy chain, the present invention is a heavy chain variable region and light chain variable regions of the heavy chain corresponding to the exchange, for example, light chain variable region of the IL-17A antibody V _L a heavy chain variable region of an antibody V _H positions interchanged with the IL-17A, since the antibody molecule has only the role of the light chain and heavy chain, thus preventing light chain with mismatches between. Compared with the existing bispecific antibodies, the bispecific antibody of the present invention is superior to the present in terms of conformational design, especially the configuration of the Fc segment, and its specificity in heterologous dimer binding. There are technologies.

The invention uses ELISA to detect the affinity of the bispecific antibody for IL-17A and TNFα, compared with the anti-TNFα, anti-IL-17A monoclonal antibody, the bispecific antibody of the invention has affinity with the antigen compared with the monoclonal antibody. There was no significant difference; the thermal stability of the bispecific antibody was detected by circular dichroism CD and found to be almost indistinguishable from the natural monoclonal antibody.

According to a specific embodiment of the present invention, the IgG hybrid type anti-TNFα and IL-17A bispecific antibody of the present invention or a biologically active fragment derived from the antibody capable of specifically binding TNFα and IL-17A, wherein The first heavy chain has the amino acid sequence of SEQ ID NO: 3, the first light chain has the amino acid sequence of SEQ ID NO: 4, and the second heavy chain has the amino acid sequence of SEQ ID NO: The second light chain has the amino acid sequence of SEQ ID NO: 6;

The bispecific antibody having SEQ ID NO: 3 to SEQ ID NO: 6 provided by the present invention has a small molecular weight (150 kDa), and the present invention provides a double compared to the bispecific antibody of more than 200 kDa provided by the prior art. Specific antibodies are closer to natural human IgG antibody molecules, making them more biocompatible.

In another aspect, the invention provides a DNA molecule encoding an IgG hybrid anti-TNFα and IL-17A bispecific antibody of the invention, or a biologically active fragment thereof, comprising an amino acid encoding SEQ ID NO: A nucleotide sequence, preferably, the nucleotide sequence is SEQ ID NO: 7.

The present invention provides a DNA molecule encoding the IgG hybrid anti-TNFα and IL-17A bispecific antibody of the present invention or a biologically active fragment thereof, comprising a nucleotide sequence encoding the amino acid having SEQ ID NO: Preferably, the nucleotide sequence is SEQ ID NO:9.

The present invention provides a DNA molecule encoding the IgG hybrid anti-TNFα and IL-17A bispecific antibody of the present invention or a biologically active fragment thereof, comprising a nucleotide sequence encoding the amino acid having SEQ ID NO: 6. Preferably, the nucleotide sequence is SEQ ID NO: 10.

In another aspect, the present invention provides HEK293F human kidney blast cells secreting the IgG hybrid anti-TNFα and IL-17A bispecific antibodies of the present invention, comprising the coding sequence of SEQ ID NO: 3, SEQ ID NO: 5 and a nucleotide sequence of the amino acid of SEQ ID NO: 6, and a nucleotide sequence encoding the amino acid having SEQ ID NO: 4, preferably, the nucleotide sequence is SEQ ID NO: 8;

Preferably, the nucleotide sequence encoding the amino acid having the SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 is integrated on a plasmid; the plasmid can be electroporated Or chemical transfection methods were transfected into 293F cell lines for expression. The invention shows that the structural expression of the antibody is correct by SDS-PAGE and western blot.

In another aspect, the present invention provides a method of producing an IgG hybrid anti-TNFα and IL-17A bispecific antibody comprising culturing the HEK293F human kidney embryo of the present invention under conditions which allow expression of the bispecific antibody The cells, and the bispecific antibodies expressed are recovered.

In another aspect, the present invention provides a pharmaceutical composition comprising the IgG hybrid anti-TNFα and IL-17A bispecific antibody of the present invention or a biologically active fragment thereof.

In another aspect, the present invention provides the IgG hybrid type anti-TNFα and IL-17A bispecific antibody of the present invention or a biologically active fragment thereof or the pharmaceutical composition prepared for the treatment of rheumatoid arthritis, Crowe Use in medicines for diseases, psoriasis, psoriasis or tumors.

In another aspect, the invention provides a kit for detecting TNFα or IL-17A levels, comprising an IgG hybrid anti-TNFα and IL-17A bispecific antibody of the invention or a biologically active fragment thereof; The kit further comprises a second antibody and an enzyme or fluorescent or radiolabel for detection, and a buffer; preferably the second antibody is an anti-antibody or anti-TNFα or IL-anti against the bispecific antibody of the present invention. 17A polyclonal antibody.

The novel stable human IgG type bispecific antibody (BiAbs) developed by the present invention is a molecule targeting two antigens, TNFα and IL-17A, which retains both the functional properties of anti-TNFα antibody binding and T cells. The additional activity of the cytokine IL-17A binding, therefore, this molecule has a better therapeutic effect on inflammatory diseases and autoimmune diseases than monoclonal antibodies directed against only one of the molecules, and the present invention proves that it can be confirmed by in vitro experiments. Blocking the damage of inflammatory factors on cells, compared with monoclonal antibodies, has obvious advantages and has broad application prospects. More importantly, the bispecific antibody of the present invention has a smaller molecular weight (150 kDa), which is closer to the natural human IgG antibody molecule than the bispecific antibody of more than 200 kDa provided by the prior art. Make it better biocompatible.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The bispecific antibody of the present invention is superior to the prior art in terms of configuration, particularly in configuration of the Fc segment, stability thereof, and specificity of heterodimer binding;

(2) Compared with the anti-TNFα or anti-IL-17A monoclonal antibody, the bispecific antibody of the present invention has no difference in affinity, stability and purity;

(3) Compared with the bispecific antibody provided by the prior art, the bispecific having SEQ ID NO: 3 to SEQ ID NO: 6 has a smaller molecular weight (150 kDa), which is closer to a natural human IgG antibody molecule. To make it more biocompatible;

(4) Compared with the existing prokaryotic expression vector, the vector of the present invention is a eukaryotic expression system, which conforms to the national FDA clinical approval drug standard.

DRAWINGS

Figure 1 is a schematic illustration of the construction of a bispecific antibody of the invention;

Figure 2 is a structural diagram of the pcDNA3.1(+)-GS-intron-IRES-CH plasmid used in Example 1;

3A to 3D are diagrams showing the results of the ELSA experiment of Example 1;

Figure 4 is a graph showing the results of the SDS-PAGE experiment of Example 1;

Figure 5 is a graph showing the results of an experiment for detecting the antagonism of a bispecific antibody against TNF-α by using L929 cells in Example 1;

6A to 6E are diagrams showing the results of real-time PCR experiments of Example 1;

Fig. 7 is a graph showing the results of experiments for measuring the T _m value of the antibody by circular dichroism CD of Example 1.

detailed description

The technical solutions of the present invention will be described in detail below with reference to the specific embodiments and the accompanying drawings. The scope of the invention. In the examples, each of the original reagent materials is commercially available, and the experimental methods not specifying the specific conditions are conventional methods and conventional conditions well known in the art, or in accordance with the conditions recommended by the instrument manufacturer.

Example 1

(1) Construction of plasmid

(1.1) Construction of recombinant vector of anti-TNFα antibody gene

(1.1.1) containing anti TNFαV _H gene (SEQ ID NO: 11) The recombinant vector pcDNA3.1 (+) - Construction of _{GS-intron-IRES-V H} -C H in

(1.1.1.1) Full-length synthesis of anti-TNFα V _H (SEQ ID NO: 11) nucleotide sequence DNA molecule, the plasmid was extracted and digested with BamHI and NheI restriction enzymes, respectively, and digested at 37 ° C, respectively. After overnight, the 450 bp fragment was recovered after digestion.

(1.1.1.2) Extraction pcDNA3.1 (+) - GS-intron -IRES-C H plasmids (construction of the plasmid shown in Figure 2, the plasmid is the C _H CH of the TNFα gene), the step (1.1 .1.1) The recovered V _H gene and the pcDNA3.1(+)-GS-intron-IRES-CH plasmid were digested with BamHI and NheI endonuclease respectively, and digested at 37 °C overnight. For the digestion of the fragment, the procedure for the recovery of the gum is described in the instruction manual of Genstar No. D205-04;

(1.1.1.3) ligation of the anti-TNFαV _H gene and the pcDNA3.1(+)-GS-intron-IRES-CH plasmid;

The pcDNA3.1(+)-GS-intron-IRES-CH plasmid recovered after digestion was taken, and the _VH gene recovered from the gel was ligated with the T4 ligase of Thermo No. EL0011;

After connecting for 1 h on a 22 ° C constant temperature metal bath, DH5α was transformed, coated with Amp ⁺ LB plate, and inverted in an incubator at 37 ° C overnight.

(1.1.1.4) PCR identification of bacterial liquid

The cells were plated on Amp-containing LB solid plate medium, and single colonies on 6 Amp ⁺ LB plates were picked and inoculated into 4 ml of Amp ⁺ LB medium. After cultured at 37 ° C for 200 hours, the colony PCR was carried out to identify positive clones and positive. Cloning indicated that the anti-TNFαV _H gene was successfully ligated to the pcDNA3.1(+)-GS-intron-IRES-CH plasmid.

(1.1.2) Construction of recombinant vector pcDNA3.1(+)-GS-intron-LC-IRES-V _H -CH containing anti-TNFαV _L- C _L gene

(1.1.2.1) Cloning of the V _L gene (SEQ ID NO: 12)

a) providing an upstream primer and a downstream primer, wherein the base sequence of the upstream primer

Base sequence of the downstream primer

b) Gene Synthesis nucleotide sequence _{V L (SEQ ID NO: 12} ) as PCR template, PCR amplified V _L gene;

After the PCR is completed, the gel recovers the band near 400 bp. For the rubber recovery step, refer to the instruction manual of Genstar No. D205-04;

(1.1.2.2) Cloning of the C _L gene (SEQ ID NO: 15)

a) provide upstream primers

And downstream primers

b) a nucleotide sequence of the whole gene synthesis of C _L gene (SEQ ID NO: 15) as PCR template DNA molecule, C _L gene was amplified by PCR;

After the PCR is completed, the strip of the 400 bp accessory is recovered by the glue. For the rubber recovery step, refer to the instruction manual of Genstar No. D205-04.

(1.1.2.3) Overlapping PCR amplification of V _L (SEQ ID NO: 12) and _CL (SEQ ID NO: 15)

Configure the amplification system as follows:

The PCR amplification procedure is:

After the end of the cycle, the following primers V _L -F and primers C _L -R were added to continue amplification;

Primer V _L -F:

Primer C _L -R:

The above primer V _L -F (20mM) 0.5μl

The above primer C _L -R (20mM) 0.5μl

The PCR amplification procedure is as follows:

After the PCR is completed, the strip of the 700 bp accessory is recovered by the glue. For the rubber recovery step, refer to the instruction manual of Genstar No. D205-04;

(1.1.2.4) Enzymatic cleavage of pcDNA3.1(+)-GS-intron-IRES-V _H- CH plasmid;

The recombinant vector pcDNA3.1(+)-GS-intron-IRES- _VH- CH plasmid containing the anti-TNFαV _H gene (SEQ ID NO: 11) obtained in the step (1.1.1) was extracted, and the step (1.1.2.3) was carried out. The recovered V _L -C _L gene and the pcDNA3.1(+)-GS-intron-IRES-V _H -CH plasmid were digested with NotI and XhoI endonuclease, respectively;

The enzyme was digested at 37 ° C for about 6 h, and the fragment was recovered by gel recovery;

(1.1.2.5) ligation of the V _L -C _L gene and the pcDNA3.1(+)-GS-intron-IRES-V _H -CH plasmid;

The pcDNA3.1(+)-GS-intron-IRES-V _H- CH plasmid recovered after digestion was taken, and the V _L -C _L gene recovered from the gel was ligated with the T4 ligase of Thermo No. EL0011;

After connecting for 1 h on a 22 ° C constant temperature metal bath, DH5α was transformed, coated with Amp ⁺ LB plate, and inverted in an incubator at 37 ° C overnight.

(1.1.2.6) PCR identification of bacterial liquid

The cells were plated on Amp-containing LB solid plate medium, and single colonies on 4 Amp ⁺ LB plates were picked and inoculated into 4 ml of Amp ⁺ LB medium. After cultured at 37 ° C for 200 hours, the colony PCR was carried out to identify positive clones and positive. Cloning means that the anti-TNFαV _L -C _L gene was successfully ligated to the pcDNA3.1(+)-GS-intron-IRES-V _H -C _H plasmid, ie, the anti-TNFα monoclonal antibody plasmid was constructed.

(1.2) Construction of recombinant vector of anti-IL-17A antibody gene

(1.2.1) Construction of recombinant vector pcDNA3.1(+)-GS-intron-IRES-V _H -CH containing anti-IL-17A V _H gene (SEQ ID NO: 20)

(1.2.1.1) The full-length synthesis of the _VH nucleotide sequence of anti-IL-17A (SEQ ID NO: 20) DNA molecule, and the plasmid was extracted and digested with BamHI and NheI restriction enzymes, respectively at 37 ° C. After digestion and overnight digestion, the 450 bp fragment was recovered by gel.

(1.2.1.2) Extraction pcDNA3.1 (+) - GS-intron -IRES-CH plasmid (construction of the plasmid shown in Figure 2, this plasmid CH C _H gene is a IL-17A), the step ( 1.2.1.1) The recovered V _H gene and the pcDNA3.1(+)-GS-intron-IRES-CH plasmid were digested with BamHI and NheI endonuclease respectively; the enzyme was digested overnight at 37 °C. The enzyme fragments are recovered; the procedure for the gum recovery is described in Genstar article number D205-04;

(1.2.1.3) ligation of the anti-IL-17A V _H gene and the pcDNA3.1(+)-GS-intron-IRES-CH plasmid;

The pcDNA3.1(+)-GS-intron-IRES-CH plasmid recovered after digestion was taken, and the _VH gene recovered from the gel was ligated with the T4 ligase of Thermo No. EL0011;

After connecting for 1 h on a 22 ° C constant temperature metal bath, DH5α was transformed, coated with Amp ⁺ LB plate, and inverted in an incubator at 37 ° C overnight.

(1.2.1.4) PCR identification of bacterial liquid

The cells were plated on Amp-containing LB solid plate medium, and single colonies on 6 Amp ⁺ LB plates were picked and inoculated into 4 ml of Amp ⁺ LB medium. After cultured at 37 ° C for 200 hours, the colony PCR was carried out to identify positive clones and positive. Cloning indicated that the anti-IL-17A V _H gene was successfully ligated to the pcDNA3.1(+)-GS-intron-IRES-CH plasmid.

(1.2.2) Construction of recombinant vector pcDNA3.1(+)-GS-intron-LC-IRES-V _H -C _H containing anti-IL-17A V _L- C _L gene

(1.2.2.1) Cloning of the V _L gene (SEQ ID NO: 21)

a) providing an upstream primer and a downstream primer, wherein the base sequence of the upstream primer

Base sequence of the downstream primer

b) Gene Synthesis V _L nucleotide sequence (SEQ ID NO: 21) as PCR template, PCR amplified V _L gene;

After the PCR is completed, the gel recovers the band near 400 bp. For the rubber recovery step, refer to the instruction manual of Genstar No. D205-04;

(1.2.2.2) Cloning of the C _L gene (SEQ ID NO: 15)

a) provide upstream primers

And downstream primers

b) C _L total gene synthesis the nucleotide sequence (SEQ ID NO: 15) PCR DNA molecule as a template, PCR amplification using the C _L gene.

After the PCR is completed, the strip of the 400 bp accessory is recovered by the glue. For the rubber recovery step, refer to the instruction manual of Genstar No. D205-04.

(1.2.2.3) Overlapping PCR amplification of V _L (SEQ ID NO: 21) and _CL (SEQ ID NO: 15)

Configure the amplification system as follows:

The PCR amplification procedure is:

After the end of the cycle, the following primers V _L -F and primers C _L -R were added to continue amplification;

The primer V _L -F has a sequence:

The primer C _L -R has a sequence:

The above primer V _L -F (20mM) 0.5μl

The above primer C _L -R (20mM) 0.5μl

The PCR amplification procedure is as follows:

After the PCR is completed, the strip of the 700 bp accessory is recovered by the glue. For the rubber recovery step, refer to the instruction manual of Genstar No. D205-04.

(1.2.2.4) Enzymatic cleavage of pcDNA3.1(+)-GS-intron-IRES-V _H- CH plasmid;

The recombinant vector pcDNA3.1(+)-GS-intron-IRES-V _H -CH plasmid containing the anti-IL-17A V _H gene (SEQ ID NO: 20) prepared in the step (1.2.1) is extracted, and the step ( 1.2.2.3) The recovered V _L -C _L gene and the pcDNA3.1(+)-GS-intron-IRES-V _H -CH plasmid were digested with NotI and XhoI endonucleases, respectively.

The enzyme was digested overnight at 37 ° C for about 6 h, and the fragment was recovered by gel recovery.

(1.2.2.5) ligation of V _L -C _L and pcDNA3.1(+)-GS-intron-IRES-V _H -CH plasmid;

The pcDNA3.1(+)-GS-intron-IRES- _VH- CH plasmid recovered after digestion was taken, and the V _L- C _L recovered from the gel was linked by T4 ligase using Thermo No. EL0011;

After connecting for 1 h on a 22 ° C constant temperature metal bath, DH5α was transformed, coated with Amp ⁺ LB plate, and inverted in an incubator at 37 ° C overnight.

(1.2.2.6) PCR identification of bacterial liquid

The cells were plated on Amp-containing LB solid plate medium, and single colonies on 4 Amp ⁺ LB plates were picked and inoculated into 4 ml of Amp ⁺ LB medium. After cultured at 37 ° C for 200 hours, the colony PCR was carried out to identify positive clones and positive. Cloning indicated that the anti-IL-17A V _L -C _L gene was successfully ligated to the pcDNA3.1(+)-GS-intron-IRES-V _H -CH plasmid, ie, the anti-IL-17A monoclonal antibody plasmid was constructed.

The plasmid of the anti-TNFα monoclonal antibody plasmid and the monoclonal antibody against IL-17A was constructed above.

(1.3) Construction of the bispecific antibody plasmid (the construction process is shown in Figure 1):

(1.3.1) Site-directed mutagenesis of CH gene fragment

(1.3.1.1) Site-directed mutagenesis of the DNA sequence at the CH fragment of the plasmid of the above-prepared anti-TNFα monoclonal antibody, the first mutation point is:

D360K

Primer pair 1

Upstream primer:

Downstream primers:

Configure the amplification system as follows:

The PCR amplification procedure is as follows:

After 16 cycles of amplification, the PCR product was cut with NEB Dpn1 enzyme, and the PCR template was removed and connected to a 37 ° C constant temperature metal bath for 1 h to transform DH5α;

At 37 ° C overnight culture, single colonies on 4 Amp ⁺ LB plates were picked and inoculated into 4 ml of Amp ⁺ LB medium, and cultured at 37 ° C for 2 h at 200 rpm and sent to the sequencing company for sequencing.

(1.3.1.2) After sequencing correctly, the second site-directed mutation D403K was performed.

The primer sequences are as follows:

Primer pair 2

Upstream primer:

Downstream primers:

The mutation process is the same as the above step (1.3.1.1);

(1.3.1.3) Site-directed mutagenesis of the CH fragment DNA sequence of the plasmid constructed to complete the above anti-IL-17A monoclonal antibody

The first mutation point is K402D

Primer pair 3

Upstream primer:

Downstream primers:

(1.3.1.4) The second mutation point is K419D

Primer pair 4

Upstream primer:

Downstream primers:

The experimental process is the same as the previous step (1.3.1.1);

(1.3.2) Exchanging the _VL (SEQ ID NO: 21) region of the plasmid having the CH gene sequence containing the region mutation of IL-17A into the _VH (SEQ ID NO: 20) region

(1.3.2.1) Performing PCR on the V _L region

a) providing an upstream primer and a downstream primer, wherein the base sequence of the upstream primer

Base sequence of the downstream primer

b) Gene Synthesis nucleotide sequence V _L PCR template, PCR amplification V _L gene.

After the PCR is completed, the gel recovers the band near 400 bp. For the rubber recovery step, refer to the instruction manual of Genstar No. D205-04;

(1.3.2.2) Fragment and plasmid digestion

a) The PCR fragments were digested with BamHI and NheI restriction enzymes respectively, and digested at 37 °C overnight to recover 400 bp fragments;

b) The anti-IL-17A plasmid mutated by the constructed CH gene sequence was digested with BamHI and NheI restriction endonuclease respectively, and the enzyme-cut fragments were recovered by enzyme digestion at 37 ° C overnight;

(1.3.2.3) The enzyme-cut fragment and the plasmid are ligated, and the T4 ligase of Thermo No. EL0011 is used for ligation;

After connecting for 1 h on a 22 ° C constant temperature metal bath, DH5α was transformed, coated with Amp ⁺ LB plate, and inverted in an incubator at 37 ° C overnight.

(1.3.2.4) PCR identification of bacterial liquid

The cells were plated on Amp-containing LB solid plate medium, and single colonies on 6 Amp ⁺ LB plates were picked and inoculated into 4 ml of Amp ⁺ LB medium. After cultured at 37 ° C for 200 hours, the colony PCR was carried out to identify positive clones and positive. Cloning indicates that the ligated fragments have been PCR amplified by PCR.

(1.3.3) the CH gene sequence containing an anti-IL-17A mutant plasmid region _{V H (SEQ ID NO: 20} ) area of the exchange to the _{V L (SEQ ID NO: 21} ) regions

(1.3.3.1) The whole gene synthesis _VH nucleotide sequence is a PCR template, and the _VH gene is amplified by PCR; an upstream primer and a downstream primer are provided, wherein the base sequence of the upstream primer

Base sequence of the downstream primer

After the PCR is completed, the gel recovers the band near 450 bp. For the rubber recovery step, refer to the instruction manual of Genstar No. D205-04;

(1.3.3.2) Cloning of the C _L (SEQ ID NO: 15) gene

All of the C _L genes in this example are identical, the light chain constant region of the antibody is C _L , and the light chain constant region is constant in the antibody. The C _L and anti-IL of the monoclonal antibody against TNFα in this example -17A C _L of a monoclonal antibody and subsequent bispecific antibodies (BsAbs) are of a C _L C _L sequence, i.e., SEQ ID NO: 15;

a) provide upstream primers

And downstream primers

b) DNA molecules the nucleotide sequence _L total gene synthesis PCR as a template C, C _L gene was amplified by PCR; After completion of the PCR, gel purification 400bp attachment strip, plastic recovery step See Cat. No. D205-04 description Genstar ;

(1.3.3.3) Then connect the V _H regions C _L together by overlapping PCR

Configure the amplification system as follows:

The PCR amplification procedure is:

After the end of the cycle, the upstream and downstream primer primers are added to continue amplification;

Primer sequence:

The PCR amplification procedure is as follows:

After the PCR is completed, the strip of the 800 bp accessory is recovered by the glue. For the rubber recovery step, refer to the instruction manual of Genstar No. D205-04;

(1.3.3.4) Double-cut the anti-IL-17A plasmid with overlapping PCR products and CH gene sequence mutations

Digestion with NotI and XhoI endonuclease, respectively;

Enzyme digestion at 37 ° C for about 6 h, after the gel recovery of the fragment;

(1.3.3.5) The overlapping PCR product recovered after digestion and the anti-IL-17A plasmid with the CH gene sequence mutation were ligated with the T4 ligase of Thermo No. EL0011, and the DH5α was transformed by plating on a constant temperature metal bath at 22 ° C for 1 h. Cloth Amp ⁺ LB plate, inverted overnight in a 37 ° C incubator;

(1.3.3.6) PCR identification and enzyme digestion identification

The cells were plated on Amp-containing LB solid plate medium, and single colonies on 4 Amp ⁺ LB plates were picked and inoculated into 4 ml of Amp ⁺ LB medium. After cultured at 37 ° C for 200 hours, the colony PCR was carried out to identify positive clones and positive. Cloning indicates that the ligated fragments have been PCR amplified by PCR.

(2) Transfecting the constructed plasmid into HEK293F cells for expression

In this embodiment, the plasmids constructed in the step (1) are transfected into the HEK293F cell line by the method of transient transfection, wherein the plasmids are respectively the plasmid of the anti-TNFα monoclonal antibody constructed above and the anti-IL- plasmid 17A monoclonal antibodies, two monoclonal antibodies were used for expression, as well as anti-TNFα construct mutant plasmid CH and _L and V _H interchangeable plasmid of anti-IL-17A mutant V good CH, wherein CH mutated anti-TNFα mutant plasmid CH and V _H and V _L plasmid interchangeable mass ratio of anti-IL17A of 1: 1, i.e. 25 ug of each plasmid, transfection 50μg total, to express bispecific antibody (BsAb);

The transfection reagent used for transient transfection is PEI25000, and the plasmid: PEI25000 transfection has a mass ratio of 1:2. The transfection step includes:

(2.1) The cells were diluted to 5×10 ⁵ /ml 24 hours before transfection;

(2.2) When transfecting, pre-heat PBS to 25-37 ° C, thaw DNA and PEI 25000;

(2.3) confirm the density and activity of the cells with a cell counter, the cell density should be between 5×10 ⁸ /ml to 1.2×10 ⁶ /ml, and the activity should be greater than 95%;

(2.4) Take 45ml of cells into a new shake flask;

(2.5) Take 5ml PBS into a 15ml centrifuge bucket, take 20μg respectively, take the prepared plasmid of step (1) and add it to the 15ml centrifuge bucket to mix;

(2.6) Add 40 μl of PEI 25000 solution with a concentration of 1 mg/ml, place in a centrifuge bucket, mix well, and vortex 3 times for 3 seconds each time;

(2.7) Place the solution at room temperature for 15 minutes; remove the cell culture flask, add the plasmid/PEI25000 mixed solution, add it while shaking, and place the cells in an incubator for shaking culture for 4 days;

(2.8) After 4 days, the cells were centrifuged and the cell supernatant was collected.

(3) ELISA test

(3.1) Cytokine TNFα and IL-17A were diluted with a coating solution, 50 ng of cytokine was added to each well, and the refrigerator was overnight at 4 ° C;

(3.2) Washing 5 times with PBST;

(3.3) plus 10g / L BSA, blocked at 37 ° C for 2h;

(3.4) Washing 5 times with PBST;

(3.5) Add three kinds of antibody supernatants, two duplicate wells per antibody, anti-TNFα, anti-IL-17A, bispecific antibody, three antibody supernatants 3 times 3 times with PBS Dilution, the highest concentration was the supernatant solution; the supernatant was not transferred into HEK293F cells as a negative control, and the BSA dilution was used as a blank control; 37 ° C for 1 h. The anti-TNFα antibody was only added to the cytokine TNFα-coated well, the anti-IL 17A antibody was only added to the cytokine IL17A-coated well, and the BsAb was added to the two cytokine-coated wells. ;

(3.6) Washing 5 times with PBST;

(3.7) adding HRP anti-human-IgG;

(3.8) Washing 5 times with PBST;

(3.9) adding TMD substrate color;

(3.10) adding 2mol/L sulfuric acid to terminate the reaction;

(3.11) measuring the absorbance of OD450nm by an enzyme-linked instrument;

The results of the positive well ELISA are shown in Figures 3A to 3D. It can be seen from Figures 3A to 3D that the constructed anti-TNFα antibody has strong binding to the antigen TNFα, and the expressed anti-IL17A antibody and the antigen IL17A have Strong binding, the constructed bispecific antibody has strong binding to both antigens, and the binding is similar to monoclonal antibodies.

(4) Purification of antibody: The collected supernatant was prepared to purify the antibody protein. All expression and purification were performed simultaneously with three antibodies, anti-TNFα, anti-IL17A and bispecific antibody (BsAb);

Purification of IgG with Protein A

(4.1) Filter the cell supernatant with a 0.45 μm filter before purification;

(4.2) Prepare 0.3 ml of Protein A into a concentrated chromatography column (BIO-RAD poly-prep chromatography columns), and wash the column once with PBS of pH 7.4;

(4.3) using the cell supernatant twice through the column;

(4.4) 2×10 mL PBS cleaning;

(4.5) Neutralization was carried out with 1 ml of sodium acetate (pH 3) and 0.25 mL of 1 M Tris-HCl pH 8.0. The concentration of acetic acid is 50 mM, and the concentration of sodium chloride is 0.15 M;

(4.6) further concentrating the concentrated antibody by ultrafiltration using a 10K ultrafiltration tube;

(5) SDS-PAGE identification: the mass fraction of the separation gel is 12%

Take an appropriate amount of sample and add 2×SDS loading buffer, add the sample tank with a micropipette, and electrophoresis at 8v/cm. When the bromophenol blue front enters the separation gel, electrophoresis is carried out at 12v/cm. After phenol blue electrophoresis to the bottom of the separation gel, the gel was taken out, stained with Coomassie blue staining solution, and the decolorizing solution was decolorized until the protein bands were clear. The results are shown in Figure 4. The left side of Figure 4 is denatured protein, and the right side is not. Denatured protein, the undenatured protein is a loading buffer in protein electrophoresis without thiol ethanol. Before protein electrophoresis, there is no boiled sample. The denatured protein is in the loading buffer. After adding thiol ethanol, the sample was boiled at 105 ° C for 10 min before loading. It can be seen from Fig. 4 that the total size of the antibody is about 150 KD, which is similar to the size of the natural antibody;

The monoclonal antibody against TNFα obtained in this embodiment is adalimumab, see US6090382;

The monoclonal antibody against IL-17A obtained in this example is secukinumab monoclonal antibody, see US20130202610;

The bispecific antibody (BsAb) obtained in this example has the amino acid sequences of SEQ ID NO: 3 to SEQ ID NO: 6.

(6) Detection of antagonism of TNF-α by bispecific antibodies using L929 cells

L929 cells were sensitive to TNF-α. The cells were induced by human recombinant TNF-α and actinomycin D. The cells died rapidly. After adding antibodies, the cell survival rate was significantly increased. Cell culture conditions: RPMI1640+10% FBS, Incubate at 37 ° C, CO ₂ concentration is 5%; when the cells are in the logarithmic growth phase, the cells are trypsinized, the density of the cells is adjusted to 1 × 10 ⁵ /mL with complete medium, and the cells are uniformly added to 96 In the well plate, 100 μl per well was cultured overnight; the cells were divided into a blank group, a bispecific antibody group (BsAb), a TNFα antibody group and an IL 17A antibody group, all of which were obtained in the present example; Recombinant TNFα and actinomycin D were combined and induced. The other four groups were first mixed with human recombinant TNFα and actinomycin D and antibody protein. The final concentration of actinomycin D was 4 μg/mL, and human recombinant TNFα was 25 ng. /mL, antibody protein concentration of 50ng / mL; placed at 37 ° C for 1h, added to L929 cells; 24h after the use of MTT method to detect cell survival rate, the results shown in Figure 5, can be seen from Figure 5 Ability of binding of sex antibody to antigen TNFα and TNFα antibody The binding activity almost always, can hinder the recombinant protein that binds human TNFα and L929 cells.

(7) Real-time PCR experiment

This experiment was used to test the effect of the bispecific antibody (BsAb), TNFα antibody and IL-17A antibody obtained in the present example on the secretion of chemokines by HT-29 cells;

HT-29 is a human colon cancer cell and has receptors for IL17A and TNFα protein. When combined with these two proteins, the content of chemokines in the cells is significantly increased. To detect the bispecific antibody pairs obtained in this example. The antagonism of these two cytokines was designed as follows:

HT-29 cell culture conditions: RPMI1640 + 10% FBS, 37 ° C, 5% CO ₂ ; When the cells were in the logarithmic growth phase, the cells were digested with trypsin, adjusted to a cell concentration of 1 × 10 ⁶ /mL, and the cells were Put into a 6-well plate, add 2 mL of cells to each well, and after cell culture for 24 hours, remove the cell culture medium and replace it with medium containing 0.5% serum. After overnight culture, divide the cells into five groups, blank group. Induction group, TNFα antibody group, IL 17A antibody group, bispecific antibody (BsAb) group, dosing treatment; blank group without any drugs, induced group added recombinant human TNFα and IL 17A cytokines, TNFα concentration was 0.5 ng / The concentration of mL and IL 17A was 50 ng/mL; the other groups were added with the above-mentioned concentrations of recombinant human cytokines, and the corresponding antibodies were also added. The concentration of the antibody was 100 ng/mL. After 12 hours of treatment, the total RNA of the cells was extracted. The expression of chemokines in HT-29 cells was detected by real-time PCR. The results obtained are shown in Figures 6A to 6E, which represent the results of chemokines CXCL1, CXCL2, CXCL6, IL8, and CCL20, respectively, from Figures 6A to 6E. It can be seen that the induction of recombinant human TNFα and IL 17A cytokines The expression levels of HT-29 cell chemokines CXCL1, CXCL2, CXCL6, IL8 and CCL20 were up-regulated, and the expression of these chemokines was observed by anti-TNFα antibody and anti-IL17A antibody and bispecific antibody. The amount was significantly reduced, and it can be seen that the effect of the bispecific antibody is better than that of the monoclonal antibody.

(8) Experiment of measuring the T _m value of antibody by circular dichroism CD

The T _m value of the antibody obtained in this example was measured by circular dichroism CD. The instrument model used in this experiment was JASCO J-815, which included the following steps:

(8.1) The sample concentration is 0.5 mg/ml, and 200 μl of the sample is added to the sample cup, and the sample cup is placed near the left side, and is clamped by the rear card slot;

(8.2) The measurement of the T _m value is carried out at a wavelength of 222 nm;

(8.3) Select the temperature/wavelength (e) interface at the software page to enter the T _m value;

Select hand tool for parameter setting

The temperature can be selected from 0 to 100 degrees and execution can be started;

The measured results are shown in Fig. 7. It can be seen from Fig. 7 that the T _m value of the anti-IL-17A antibody is about 82 ° C, and the T _m value of the bispecific antibody and the anti-TNFα antibody is similar, about 68 ° C. Around, it was found that its thermal stability is almost the same as that of natural monoclonal antibodies.

Claims (12)

1. An IgG hybrid anti-TNFα and IL-17A bispecific antibody or a biologically active fragment derived from the bispecific antibody capable of specifically binding to TNFα and IL-17A, the bispecific antibody having a human IgG type a first heavy chain and a first light chain for specifically binding to TNFα, and a second heavy chain and a second light chain for specifically binding IL-17A by a human IgG type;

   Wherein the variable region in the first heavy chain is a variable region of a light chain in an antibody of human IgG-type TNFα; the variable region in the first light chain is an antibody to the human IgG-type TNFα Variable region in the heavy chain; and/or

   The variable region in the second heavy chain is a variable region in the light chain of a human IgG type IL-17A antibody; the variable region in the second light chain is an antibody to the human IgG type IL-17A Variable region in the heavy chain;

   The constant regions in the first heavy chain and the second heavy chain are bonded to each other by enhanced electrostatic interaction and/or hydrophobic interaction.
2. The IgG hybrid anti-TNFα and IL-17A bispecific antibody according to claim 1 or a biologically active fragment derived from the antibody capable of specifically binding TNFα and IL-17A, wherein the enhanced electrostatic effect is The K402D and/or K419D mutation is carried out in the constant region of the heavy chain of the antibody of human IgG-type IL-17A by subjecting the constant region in the heavy chain of the antibody of human IgG-type TNFα to D360K and/or D403K mutation. Achieved.
3. The IgG hybrid anti-TNFα and IL-17A bispecific antibody according to claim 2 or a biologically active fragment derived from the antibody capable of specifically binding TNFα and IL-17A, wherein the human IgG type TNFα The heavy chain of the antibody is SEQ ID NO: 1, and the amino acids 360 and 403 in the heavy chain of the human IgG type TNFα antibody are replaced by Asp to Lys; the human IgG type IL-17A antibody The heavy chain is SEQ ID NO: 2, and positions 402, 419 in the heavy chain of the antibody of human IgG type IL-17A are replaced by Lys to Asp.
4. The IgG hybrid anti-TNFα and IL-17A bispecific antibody according to claim 1 or a biologically active fragment derived from the antibody capable of specifically binding TNFα and IL-17A, wherein the first heavy chain has An amino acid sequence of SEQ ID NO: 3, wherein the first light chain has the amino acid sequence of SEQ ID NO: 4, the second heavy chain has the amino acid sequence of SEQ ID NO: 5, and the second light chain has The amino acid sequence of SEQ ID NO: 6.
5. A DNA molecule encoding the IgG hybrid anti-TNFα and IL-17A bispecific antibody of claim 1, comprising a nucleotide sequence encoding an amino acid having SEQ ID NO: 3, preferably, the nucleoside The acid sequence is SEQ ID NO: 7.
6. A DNA molecule encoding the IgG hybrid anti-TNFα and IL-17A bispecific antibody of claim 1, comprising a nucleotide sequence encoding an amino acid having SEQ ID NO: 5, preferably, the nucleoside The acid sequence is SEQ ID NO:9.
7. A DNA molecule encoding the IgG hybrid anti-TNFα and IL-17A bispecific antibody of claim 1, comprising a nucleotide sequence encoding an amino acid having SEQ ID NO: 6, preferably, the nucleoside The acid sequence is SEQ ID NO: 10.
8. HEK293F human kidney embryo cell secreting the IgG hybrid anti-TNFα and IL-17A bispecific antibody according to claim 1, comprising the DNA molecule according to claim 5, the DNA molecule according to claim 6, and the claims 7 a DNA molecule, and a nucleotide sequence encoding the amino acid having SEQ ID NO: 4, preferably, the nucleotide sequence is SEQ ID NO: 8;

   Preferably, the nucleotide sequence encoding the amino acid having the SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 is integrated on a plasmid; the plasmid can be chemically transfected Methods Transfection into HEK293F cells for expression.
9. A method for producing an IgG hybrid type anti-TNFα and IL-17A bispecific antibody, comprising culturing the HEK293F human kidney embryo cell of claim 8 under conditions which allow expression of the bispecific antibody, and recovering the expressed Bispecific antibody.
10. A pharmaceutical composition comprising the IgG hybrid anti-TNFα and IL-17A bispecific antibody according to any one of claims 1 to 4 or an antibody capable of specifically binding to TNFα and IL-17A Biologically active fragment.
11. The IgG hybrid anti-TNFα and IL-17A bispecific antibody according to any one of claims 1 to 4, or a biologically active fragment derived from the antibody capable of specifically binding TNFα and IL-17A or according to claim 10. Use of the pharmaceutical composition for the preparation of a medicament for the treatment of rheumatoid arthritis, Crohn's disease, psoriasis, psoriasis or tumors.
12. A kit for detecting the level of TNFα or IL-17A, comprising the IgG hybrid type anti-TNFα and IL-17A bispecific antibody according to any one of claims 1 to 4 or capable of specificity derived from the antibody Combining biologically active fragments of TNFα and IL-17A; preferably the kit further comprises a second antibody and an enzyme or fluorescent or radioactive label for detection, and a buffer; preferably the second antibody is resistant to claim 1 The anti-antibody of the bispecific antibody or the polyclonal antibody against TNFα or IL-17A according to any one of the above.

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