CN110903393B

CN110903393B - Polypeptide capable of binding IL6R alpha and application thereof

Info

Publication number: CN110903393B
Application number: CN201911298163.1A
Authority: CN
Inventors: 黄碧莲; 吴喜林; 吴稚伟; 瓦卡斯·那瓦兹
Original assignee: Yuandaolong Suzhou Medical Technology Co ltd
Current assignee: Yuandaolong Suzhou Medical Technology Co ltd
Priority date: 2019-12-17
Filing date: 2019-12-17
Publication date: 2021-08-13
Anticipated expiration: 2039-12-17
Also published as: CN110903393A

Abstract

The invention relates to a polypeptide capable of binding IL6R alpha, which comprises 3 complementarity determining regions CDR1-3, wherein the sequence of CDR1 is or comprises the sequence shown in SEQ ID NO. 1, the sequence of CDR2 is or comprises one of the sequences shown in SEQ ID NO. 2-4, and the sequence of CDR3 is or comprises one of the sequences shown in SEQ ID NO. 5-7. According to the invention, a nano antibody VHH specifically combined with IL6R alpha is screened by preparing IL6R alpha protein, immunizing alpaca, utilizing a platform technology of phage library display nano monoclonal antibody and the like, the CDR sequence of the nano antibody is identified, and a humanized nano antibody is constructed; and simultaneously evaluating the function of the humanized antibody for blocking the combination of IL6 and IL6R by using an in vitro functional test. The invention provides a potential nano-antibody new drug for clinical treatment of autoimmune diseases, inflammatory diseases or tumor diseases such as rheumatoid arthritis and the like related to IL6 and a ligand thereof.

Description

Polypeptide capable of binding IL6R alpha and application thereof

Technical Field

The invention relates to the field of biomedicine. More particularly, it relates to a polypeptide capable of binding human IL6R alpha, and the application of said polypeptide in the therapeutic medicine for autoimmune diseases, inflammatory diseases or tumor diseases, such as Rheumatoid Arthritis (RA).

Background

IL-6 is a pleiotropic proinflammatory cytokine with a variety of biological activities, including mediating inflammatory responses, immune responses, and the like. IL-6 is highly expressed in a number of inflammatory diseases, such as rheumatoid arthritis, Systemic Lupus Erythematosus (SLE), Crohn's disease, psoriasis, and the like. In recent years there has been increasing evidence that IL-6/IL-6R is involved in the development and prognosis of a variety of diseases, such as inflammatory diseases, autoimmune diseases and tumours. IL-6 is reported to be combined with an IL-6 receptor (IL-6R), activate gp130, trigger JAK-STAT signaling pathway, express inflammation-related factors, finally cause some inflammatory diseases and autoimmune diseases, and is also related to the occurrence and development of tumors. In the above reaction process, IL-6 receptor alpha chain IL6R alpha plays an important role, is IL-6 binding site.

In 1993, a novel natural antibody derived from camelidae was found. The antibody naturally lacks a light chain and consists only of a heavy chain comprising two constant regions (CH2 and CH3), a hinge region and a heavy chain Variable region (VHH, i.e., antigen binding site) with a relative molecular mass of about 13KDa, which is only 1/10 of conventional antibodies, and with a molecular height and diameter at the nanometer level, is the smallest functional antibody fragment currently available, and thus is also referred to as Nanobody (Nb). Because the nano monoclonal antibody has the characteristics of high stability (not degraded at 90 ℃), high affinity, homology of more than 80 percent with a human antibody, low toxicity and immunogenicity and the like, the nano monoclonal antibody is widely applied to the research and development of immunodiagnosis kits, the research and development of imaging, and the research and development of antibody drugs aiming at the fields of tumors, inflammations, infectious diseases, nervous system diseases and the like.

The only antibody currently used in clinical application of human IL6R is Tocilizumab, which is approved by the U.S. food and drug administration for the treatment of rheumatoid arthritis and is suitable for adult moderate-to-severe rheumatoid arthritis patients who are ineffectual to use other approved drugs. In the case where there is only one IL6R antibody worldwide, the global market is open. Therefore, the development of new IL6R antibody drugs is urgently required.

Disclosure of Invention

The alpaca source nanometer monoclonal antibody and the VHH thereof are obtained by immunizing alpaca with antigen and are used for patients with autoimmune diseases. Based on these studies, the present invention provides a polypeptide capable of binding IL6R α, comprising 3 CDRs 1-3, wherein CDR1 sequence is one of the sequences shown in SEQ ID NO. 1, CDR2 sequence is or comprises one of the sequences shown in SEQ ID NO. 2-4, and CDR3 sequence is or comprises one of the sequences shown in SEQ ID NO. 5-7.

In a specific embodiment, the polypeptide further comprises 4 framework regions FR1-4, said FR1-4 being staggered with respect to said CDR 1-3. For example, the FR1-4 sequence can be designed as shown in SEQ ID NOS: 8-11 (sources of alpaca), but the scope of the present invention is not limited thereto, and the framework region FR1-4 sequence can also be humanized as shown in SEQ ID NOS: 12-15. The specific recognition and binding ability of an antibody is mainly determined by the CDR region sequences, and the FR sequences have little influence and can be designed according to species, which is well known in the art. For example, FR region sequences of human, murine or camelid origin may be designed to join the above CDRs to form a polypeptide or domain which binds human IL6R α.

In a preferred embodiment, the polypeptide is a monoclonal antibody.

In a preferred embodiment, the polypeptide is VHH.

In a preferred embodiment, the polypeptide is a VHH of alpaca origin or a humanized VHH.

In one embodiment, the CDR sequences of the polypeptides are as follows:

I) the sequence of CDR1 is SEQ ID NO. 1, the sequence of CDR2 is SEQ ID NO. 16, and the amino acid sequence shown in SEQ ID NO:6, X at position 4 represents serine or asparagine, and X at position 6 represents glycine or aspartic acid; and is

II) CDR3 has a sequence selected from SEQ ID NO 5-7.

Preferably, the CDR sequences of the polypeptides are as follows:

the sequence of CDR1 is SEQ ID NO 1, the sequence of CDR2 is SEQ ID NO 2 and the sequence of CDR3 is SEQ ID NO 5; or

The sequence of CDR1 is SEQ ID NO 1, the sequence of CDR2 is SEQ ID NO 3 and the sequence of CDR3 is SEQ ID NO 6; or

The sequence of CDR1 is SEQ ID NO. 1, the sequence of CDR2 is SEQ ID NO. 4 and the sequence of CDR3 is SEQ ID NO. 7; or

The invention also provides application of the polypeptide in detecting cell surface IL6R alpha.

The invention also provides the application of the polypeptide in preparing the medicine for treating autoimmune diseases.

The present invention also provides the nucleic acid encoding sequence of the polypeptide and its application in gene treating medicine.

In one embodiment, the nucleic acid coding sequence is a DNA coding sequence or an RNA coding sequence.

According to the invention, a nano antibody VHH specifically combined with IL6R alpha is screened by preparing IL6R alpha protein, immunizing alpaca, utilizing a platform technology of phage library display nano monoclonal antibody and the like, a CDR sequence of the nano antibody VHH is identified, and a humanized VHH-huFc1(I6RNB) is constructed; in vitro functional assays were also used to assess the ability of I6RNB to block binding of IL6 and IL 6R. The invention provides a potential nano-antibody new drug for clinical treatment of autoimmune diseases, inflammatory diseases or tumor diseases such as rheumatoid arthritis and the like related to IL6 and a ligand thereof.

Drawings

FIG. 1 is a test curve of antiserum titer of sIL6R alpha-rFc one week after 3rd immunization of alpaca;

FIG. 2 is a flow assay of sIL6R α -rFc immune alpaca serum binding to U937 cells expressing IL6R α. NC control is nonimmune alpaca serum;

FIG. 3 is an electrophoretogram of PCR products amplified using sIL6R α -VHH phage antibody library as a template;

FIG. 4 shows the panning identification of the sIL6R alpha-VHH phage antibody library, where A is a phage library against

ELISA detection statistical profile after panning of sIL6R a protein; b is a second wheel (2)^nd) And a third wheel (3)^rd) Respectively selecting 48 clones and 38 clones from the panned phage antibody library to carry out phage ELISA detection statistical chart;

FIG. 5 is a statistical ELISA assay for prokaryotically expressed VHH antibodies, each dot representing a clone, with OD450 for sIL6R α/OD 450 for blank on the ordinate, and a positive ratio greater than 5.0 being defined;

FIG. 6 is a flow assay of prokaryotically expressed VHH antibodies blocking the binding of IL6 to IL6R α on Jurkat cells.

Detailed Description

1. Preparation of immunogens

According to the sequence and gene sequence information of human IL6R alpha protein on NCBI website, polypeptide sIL6R alpha capable of effectively inducing alpaca to generate specific antibody aiming at human IL6R alpha protein is analyzed and designed, and His-tag (sIL6R alpha-His) or rabbit Fc (sIL6R alpha-rFc) is connected at the C terminal for subsequent purification and detection.

2. Preparation of alpaca immune and antiserum

Priming alpacas with an emulsified mixture of 250 μ g of sIL6R α -rFc protein and 250 μ l of Freund's complete adjuvant, boosting 2 times with sIL6R α -rFc protein and 250 μ l of Freund's incomplete adjuvant on days 14 and 28, and collecting blood to detect antiserum titer 1 week after 2 and 3 weeks of immunization; after 1 week of the 3rd immunization, 100ml of blood was collected for phage antibody library construction.

Antiserum titers were measured by ELISA, assay plates were coated with sIL6R α -his protein at a concentration of 0.5 μ g/ml, and a gradient of diluted antiserum (control was pre-immune alpaca serum) was added to each well, incubated at 37 ℃ for 1.5h, washed 2 times, and 1: 10000 diluted second antibody of horse radish peroxidase labeled Goat anti-Llama IgG (H + L) is incubated for 1H at 37 ℃, after washing for 4-6 times, 100 mu L of TMB substrate is added, incubation is carried out for 10min at 37 ℃, and 50 mu L of 0.2M H is added₂SO₄The reaction was stopped and the OD450 nm was measured. ELISA assay serum titers were specified at the highest dilution of OD450 above 2.1-fold of blank and greater than 0.2.

As shown in FIGS. 1 and 2, the antiserum titer of 3-immunization was 1.09X 10⁶. It can be seen that the antigen can induce camels to produce high titers of antiserum specific for the sIL6R a protein.

Construction and panning of VHH phage library

Collecting 100ml of immunized alpaca peripheral blood, separating by using lymphocyte separation liquid (GE Ficoll-Paque Plus) to obtain camel PBMC, extracting RNA according to a TRIzol operation manual, inverting by using oligo (dT) into cDNA, cloning the camel VHH gene to phagemid plasmid by using techniques such as primer amplification, molecular cloning and the like, and transforming TG1 bacteria to obtain the VHH phage library. To further identify whether the sIL6R alpha-VHH phage libraryThe construction was successful, and the VHH target gene of immune sIL6R alpha alpaca was amplified by PCR, and it was found that the target band was 500bp, and the size was as expected (FIG. 3), indicating that the sIL6R alpha-VHH phage antibody library contains VHH gene. Selecting 16 clones for sequencing, wherein the sequencing result shows that the diversity of the sequenced sequence is 93.75%; the alignment results show that the most of the different sequences are in the CDR binding region. Through detection, the construction obtains an sIL6R alpha-VHH phage antibody library, and the library capacity is 1.1 multiplied by 10⁹The positive rate was 100%, the sequence Diversity (Diversity) was 93.75%, and the effective insertion rate (In frame rate) was 89%.

The phage antibody library was recovered from VHH-phagemid transformed bacteria with the help of M13KO7 helper phage and precipitated with PEG/NaCl. Phage antibody libraries were enriched three times with 50. mu.g/ml of sIL 6R. alpha. -His protein coating. And (3) carrying out elution, transformation, plate coating and monoclonal picking on the enriched phage, carrying out binding identification on the phage and sIL6R alpha protein ELISA, sequencing the clone with the binding reading value T/B >5, cloning to an expression vector pVAX1, and transfecting 293F cells to express to produce the nano monoclonal antibody.

The panning library was tested for binding to sIL6R protein. The phage ELISA results showed that the reading values of the binding between the sIL6R-VHH phage library and the sIL6R protein before enrichment were 0.14, and the reading values of the phage library after one, two and three rounds of enrichment were 0.37, 2.70 and 3.44 respectively (FIG. 4A). To further verify the positive phage rate of sIL6R-VHH protein binding in the enriched library, 48 and 38 clones were selected from the enriched libraries in round 2 and round 3, respectively, for single phage ELISA detection. The results showed that 66.7% of the individual phage clones were positive in the library round 2 and 97.4% of the phage clones were positive in the library round 3, and the mean reading for binding was around 3.0 (FIG. 4B), and the high binding sIL6R-VHH phage library was successfully enriched by protein panning with sIL 6R.

Construction of VHH prokaryotic expression library and VHH expression

PCR amplification of the enriched 2nd-sIL6R-VHH and 3rd-sIL6R-VHH phage antibody libraries after the two and three rounds of panning described above; obtaining and purifying all VHH gene fragments in an antibody library, cloning the VHH gene fragments to a prokaryotic expression vector, converting an SS320 strain, and constructing a prokaryotic expression antibody library of the VHH; and (3) coating a plate with the prokaryotic expression antibody library, culturing overnight, randomly selecting 1000 monoclonal colonies the next day, inducing and expressing the antibody supernatant by using IPTG (isopropyl-beta-thiogalactoside), and performing ELISA (enzyme-linked immuno sorbent assay) combination detection on the antibody supernatant and the sIL6R protein.

The results show that there was bacterial supernatant binding to sIL6R protein while not binding to the blank, and that sIL6R bound reads/blank reads greater than 5.0 (fig. 5). Sequencing and aligning the sequences, and removing repeated sequences to finally obtain 210 VHH antibody sequences. Of these 3 VHH antibodies had very high binding values for IL-6R α protein (Table 1).

TABLE 13 binding values of VHH antibodies to sIL6R protein and the CDR sequences thereof

Experiment of VHH antibody blocking binding of IL6 to cell surface IL6R alpha

Jurkat cells, a human acute T lymphocyte cell line, surface-expressed IL6R α, were tested for the ability of antibodies to block the binding of IL6 to cell surface IL6R α by incubating a humanized form of the three VHH antibodies described above, antibody I6RNB, in admixture with Jurkat cells. The method comprises the following steps:

1) jurkat cells were co-incubated with antibody supernatant for 1 hour at 4 ℃;

2) washing the cells with 0.5% PBSF for three times, adding IL6-rFC protein, 2 μ g/ml, and incubating at 4 deg.C for 30 min;

3) washing the cells for three times by 0.5% PBSF, adding Alexa Fluor 488 goat anti-rabbit IgG, and incubating for 30min at 4 ℃;

4) washing the cells for three times by 0.5% PBSF, and then carrying out on-machine detection on the resuspended cells;

control is no antibody incubation, only IL6-rFC protein incubation.

The results are shown in figure 6, and these three humanized VHH antibodies can block to some extent the binding of IL6 to cell surface IL6R α (figure 6).

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<110> Source daolong (Suzhou) medical science and technology, Inc

<120> polypeptide capable of binding IL6R alpha and application thereof

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Ala Ser Gly Phe Thr Leu Asp Tyr Tyr Ala

1 5 10

<210> 2

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Ile Ser Ser Asn Asp Asp Ser Thr

1 5

<210> 3

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Ile Ser Ser Asn Asp Gly Ser Thr

1 5

<210> 4

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Ile Ser Ser Ser Asp Gly Ser Thr

1 5

<210> 5

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Ala Ala Thr Met Leu Thr Tyr Gln Ala Met Gly Val Pro Val Gln Tyr

1 5 10 15

Tyr Glu Tyr Asp Tyr

20

<210> 6

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Ala Thr Val Leu Leu Thr Glu Arg Ala Val Gly Val Pro Val Met Asp

1 5 10 15

Tyr Glu Tyr Asp Tyr

20

<210> 7

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Ala Gly Val Leu Thr Thr Ala Gln Ala Met Gly Val Pro Arg Phe Gly

1 5 10 15

Tyr Glu Tyr Asp Tyr

20

<210> 8

<211> 25

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 9

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ser

1 5 10 15

Cys

<210> 10

<211> 38

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Tyr Tyr Ala Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn

1 5 10 15

Ala Lys Ser Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 11

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

1 5 10

<210> 12

<211> 25

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Gln Val Arg Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Glu

1 5 10 15

Thr Leu Arg Leu Ser Cys Thr Ala Ser

20 25

<210> 13

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Met Gly Trp Tyr Arg Gln Gly Pro Gly Asn Glu Cys Glu Met Val Ala

1 5 10 15

Tyr

<210> 14

<211> 36

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Ala Asp Ser Thr Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys

1 5 10 15

His Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly

20 25 30

Val Tyr Tyr Cys

35

<210> 15

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Gly Gln Gly Thr Arg Val Thr Val Ser Ser

1 5 10

<210> 16

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Ile Ser Ser Xaa Asp Xaa Ser Thr

1 5

Claims

1. A nanobody capable of binding IL6R alpha, comprising 3 CDRs 1-3,

the sequence of the CDR1 is shown as SEQ ID NO. 1, the sequence of the CDR2 is shown as SEQ ID NO. 2 and the sequence of the CDR3 is shown as SEQ ID NO. 5; or

The sequence of the CDR1 is shown as SEQ ID NO. 1, the sequence of the CDR2 is shown as SEQ ID NO. 3 and the sequence of the CDR3 is shown as SEQ ID NO. 6; or

The CDR1 sequence is shown in SEQ ID NO. 1, the CDR2 sequence is shown in SEQ ID NO. 4 and the CDR3 sequence is shown in SEQ ID NO. 7.

2. The nanobody of claim 1, further comprising 4 framework regions FR1-4, wherein the FR1-4 is sequentially staggered from the CDR 1-3.

3. Nanobody according to claim 2, characterized in that it is a VHH of alpaca origin or a humanized VHH.

4. Use of the nanobody of any one of claims 1 to 3 for the detection of cell surface IL6R α.

5. Use of the nanobody of any one of claims 1 to 3 for the preparation of a medicament for the treatment of rheumatoid arthritis.

6. A nucleic acid encoding the nanobody of any one of claims 1 to 3.