CN115925949A - Preparation and application of anti-IGF 1R single-domain antibody, fusion protein thereof and humanized single-domain antibody - Google Patents

Abstract

The invention relates to an anti-IGF 1R single domain antibody, a fusion protein thereof and preparation and application of a humanized single domain antibody. The single domain antibody comprises 3 unique sequences, the corresponding clone numbers are 1D7, 3B9 and 3G6, and each of the three sequences consists of a CDR region and a variable region; 1D7 is shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively; the amino acid sequence of the variable region is SEQ ID NO.10. The amino acid sequences of the CDR regions of the 3B9 are respectively shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6; the amino acid sequence of the variable region is SEQ ID NO.11; the CDR region amino acid sequences of 3G6 are respectively shown as SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO. 9; the amino acid sequence of the variable region is SEQ ID NO.12. The invention screens and obtains a group of anti-IGF 1R single-domain antibodies with high affinity and biological function inhibition activity, which can completely block the combination of IGF1R and IGF1 and also can block the specific combination of the combination of IGF1R and IGF 2.

Description

Preparation and application of anti-IGF 1R single-domain antibody, fusion protein thereof and humanized single-domain antibody

Technical Field

The invention relates to the technical field of biomedicine, in particular to a single-domain antibody, and especially relates to an IGF1R (insulin-like growth factor) resistant single-domain antibody, a fusion protein thereof and preparation and application of a humanized single-domain antibody.

Background

Thyroid Eye Disease (TED), also known as hyperthyroid exophthalmia, is a serious, progressive, vision-threatening autoimmune disease, the most common orbital disease in adults. The pathogenesis of TED is not completely clear, and at present, the TED is mainly considered to be caused by hyperthyroidism and excessive metabolism, wherein the activation of a thyrotropin receptor (TSHR) and insulin-like growth factor receptor-1 (IGF 1R) complex is a very important link of TED, and can cause abnormal immunoproliferation reaction in orbits, so that hypertrophy of extraocular muscles and increase of intraorbital fat tissues are caused, and various clinical manifestations such as exophthalmos, diplopia, blurred vision, pain, compressive optic neuropathy and the like are caused.

An insulin-like growth factor 1receptor (IGF-1R) belongs to a tyrosine protein kinase receptor family, is a cell surface transmembrane protein, can be activated by IGF-1 and IGF-2 (both insulin growth factors), and can be related to malignant tumors such as multiple sclerosis, crohn's disease, pulmonary fibrosis and the like and autoimmune diseases due to overexpression. IgG related to IGF-1R can activate IGF-1R positive orbital fibroblasts from thyroid related eye disease patients, so that activation of Akt/FRAP/mTOR/P70s6k channel induces the expression of interleukin-16 and the factors (regulated activated normal T cell expressed and secreted) for activating normal T cell expression and secretion, promotes the synthesis of T cell chemotactic factors, and causes inflammatory infiltration of T lymphocytes and the production of hyaluronic acid.

The key to the therapeutic role played by IGF1R antibodies is the ability to block the binding of IGF1R to its ligand, IGF 1. In 2020, the U.S. FDA approved the anti-IGF 1R antibody teprtumumab (trade name Tepezza) for the treatment of thyroid eye disease. It is the only approved medicine for treating thyroid eye disease in the world. Clinical trials have shown that the patients receiving teprotumumab treatment experienced a significant reduction in the outbreak of the eyeball. The phase III clinical research result shows that the research reaches the main endpoint and the tolerance is good. However, the marketed teproumumab cannot completely block the binding of IGF1R and IGF1, and the antibody of the present invention can completely block the binding of IGF1R and IGF1, and also can block the binding of IGF1R and IGF2, has higher affinity and biological function inhibitory activity than teproumumab, can act under lower dosage conditions, and has expected clinical efficacy superior to that of teproumumab. Therefore, there is a great need to develop novel therapeutic antibodies for IGF-1R for the treatment of this disease.

Disclosure of Invention

The technical problem to be solved is as follows: aiming at the defects of the prior art, the invention provides a single-domain antibody capable of binding IGF-1R, a fusion protein and a preparation method of a humanized single-domain antibody thereof, and provides a new way for developing novel treatments for related diseases mediated by IGF1R.

The technical scheme is as follows: a set of IGF1R single domain antibodies comprising 3 unique sequences, corresponding clone numbers 1D7, 3B9 and 3G6, each consisting of a CDR region and a variable region.

Furthermore, the amino acid sequences of the CDR regions in the unique sequence 1D7 are respectively shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, and the amino acid sequence of the variable region is SEQ ID NO.10.

Furthermore, the amino acid sequences of the CDR regions in the unique sequence 3B9 are respectively shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, and the amino acid sequence of the variable region is SEQ ID NO.11.

Furthermore, the amino acid sequences of the CDR regions in the unique sequence 3G6 are respectively shown as SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9, and the amino acid sequence of the variable region is SEQ ID NO.12.

The fusion protein is obtained by recombining the IGF1R single domain antibody with a pCDNA3.1 recombinant vector of a nucleic acid sequence of human IgG1 Fc (the C end of VHH is directly connected with the N end of a human Fc hinge region).

Furthermore, the sequence of the Fc region of the human IgG1 is shown in SEQ ID NO. 13; the sequence of the single-domain antibody-Fc fusion protein is shown in SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO. 16.

Carrying out humanized transformation on a variable region of a humanized IGF1R single-domain antibody 3B9 to obtain an 8 humanized antibody 3B9-hz1, a variable region; 3B9-hz2, variable domain; 3B9-hz3, variable domain; 3B9-hz4, variable domain; 3B9-hz5, variable domain; 3B9-hz6, variable domain; 3B9-hz7, variable domain; and 3B9-hz8, variable domain; the amino acid sequences are respectively shown in SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23 and SEQ ID NO. 24.

The IGF1R single domain antibody, the fusion protein and the human-derived IGF1R single domain antibody are applied to the medicines for relevant diseases induced by the overexpression of IGF1R.

Further, the related diseases include thyroid eye disease and the like.

The preparation method of the anti-IGF 1R single-domain antibody, the fusion protein and the humanized single-domain antibody thereof comprises the following steps:

s1: preparation and screening method of IGF1R single-domain antibodies

(1) Immunization of Alpaca-the immunization of Alpaca (Alpaca) with recombinant human IGF1R protein (purchased from Beijing Yiqiao Shenzhou, cat # 10164-H08H);

(2) Construction of immune library: using M13KO7 to assist phage infection, and preparing a phage display library;

(3) Panning of phage library: the single domain antibody targeting IGF1R was panned by solid phase affinity screening, three rounds of panning were performed, the DNA sequence obtained by sequencing was translated into an amino acid sequence and then compared, the repeat sequence or similar sequence differing by 1 amino acid in CDR3 was removed, and unique sequences were obtained, corresponding clone numbers 1D7, 3B9 and 3G6 (the amino acid sequences of CDR regions and variable region amino acid sequences thereof are shown in table 1 below, wherein the CDR regions were divided using the Kabat method).

S2: preparation method of fusion protein

And (3) carrying out pCDNA3.1 recombination on the single-domain antibody in the S1 and a nucleic acid sequence of human IgG1 Fc (the C end of the VHH is directly connected with the N end of a human Fc hinge region) to obtain the single-domain antibody-Fc fusion protein.

S3: single domain antibody humanization methods

The humanized antibody sequences of 3B9-hz1 to 3B9-hz8 were designed (the sequences are shown in Table 3). These humanized sequences were constructed on human IgG1 Fc (sequence shown in SEQ ID NO: 13) to form a single domain antibody-Fc fusion protein. These humanized single domain antibody-Fc fusion proteins were expressed and purified by transient transfection of the Expi293 mammalian expression system.

Has the beneficial effects that:

the invention obtains the anti-IGF 1R single-domain antibody with high affinity by screening through animal immunization and phage display technologies, can block the combination of IGF1R and ligand IGF1 or IGF2 thereof, and has an effect superior to the expected clinical curative effect of Teprotumumab possibly under the condition of lower dosage.

Drawings

FIG. 1 shows ELISA detection of binding of crude single domain antibody extracts diluted at different times to human IGF1R.

FIG. 2 is a graph of the effect of competitive ELISA assays on IGF1-IGF1R interaction.

FIG. 3 is a single domain antibody-Fc fusion protein SDS-PAGE picture.

FIG. 4 is a graph of the effect of competitive ELISA assays on IGF1-IGF1R interaction.

FIG. 5 is a graph of the effect of competitive ELISA assays on IGF2-IGF1R interaction.

FIG. 6 shows the binding of the test substance to IGF1R of human, mouse or monkey.

FIG. 7 is a graph of the effect of competitive ELISA assays on IGF1-IGF1R interaction.

FIG. 8 shows that 3B9-hz8-Fc significantly inhibited IGF 1-induced proliferation of MCF7 cells.

Detailed Description

In order to make the objects, technical solutions and effects of the present invention clearer and clearer, the present invention will be further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Example 1 preparation and screening of IGF 1R-targeting Single Domain antibodies

(1) Immunization of alpaca

A recombinant human IGF1R protein (purchased from Beijing Yiqiao Shenzhou, cat # 10164-H08H) was used to immunize llama (Alpaca). The immunization is carried out for 4 times, the dosage of each immunization is 0.5mg, and the immunization interval is two weeks. Complete adjuvant is used in the first immunization, and incomplete adjuvant is used in the rest.

(2) Immune library construction

One week after the fourth immunization, 50ml of alpaca peripheral blood was collected, lymphocytes were obtained by separation using a lymphocyte separation medium (solibao, P8610), and then RNA extraction was performed using an RNA extraction reagent (TaKaRa, 9109) according to the procedure described. Then, reverse transcription is carried out on the RNA by using a reverse transcription kit (Thermo Scientific, K1622) to prepare cDNA; then amplifying DNA containing single domain antibody variable region gene by using a nested PCR method, carrying out enzyme digestion on the DNA by using Sfi I, and carrying out T on the DNA and a display carrier subjected to enzyme digestion by using Sfi I ₄ DNA ligase ligation; and (3) after purifying the connecting product, electrically transferring TG1 competent cells, and infecting the competent cells by using M13KO7 helper phage to prepare a phage display library. The library capacity of TG1 after electrotransformation is 2.4 multiplied by 10 measured by a plate gradient dilution method ⁹ cfu。

(3) Panning of phage libraries

And panning the IGF1R targeted single-domain antibody by a solid-phase affinity screening method, and performing three rounds of panning. IGF1R protein was coated on high adsorption 96-well plates (first, second and third rounds of coating at 500ng, 200ng and 50ng per well, respectively) at 4 ℃ overnight. PBST containing 3% bovine serum albumin was then added as a blocking solution and blocked at 37 ℃ for 1 hour. Removing the blocking solution, adding the library phage (3X 10) of (2) ¹² cfu). After incubation for 1 hour at 37 ℃ for binding, the bound or less bound phage were removed by washing 10 times with PBST wash containing 0.1% Tween-20 for 2min with shaking. Finally, 100. Mu.L of glycine-hydrochloride buffer (pH 2.2) was added to dissociate the specific phage, and after gentle shaking for 10 minutes, the eluate containing the specific phage was collected and neutralized with Tris solution.

Infecting the eluate with E.coli TG1 in an exponential phase, performing gradient dilution on a small amount of infected E.coli TG1, uniformly coating an LB solid culture medium (plate dilution method) with a proper amount of gradient dilution solution, counting plates after 12-14 hours, and calculating the elution amount of phage gel; the remaining amplifications yielded a specific phage enrichment library for the next round of screening. Plates for determination of eluate titer from the second and third rounds described aboveEach of 200 clones was randomly picked into 96-well plates and cultured overnight in 2YT medium containing Carb and M13KO7 helper phage. The next day, the plates were centrifuged at 3000rpm and the supernatants were taken for phage ELISA. The method comprises the following steps: coating recombinant human IGF1R protein (coating concentration 2 mug/mL), sealing with PBST containing 3% bovine serum albumin, adding phage supernatant to be identified, binding at 37 deg.C for 1 hr, washing, adding anti-M13 antibody (product number 11973-MM 05T-H) coupled with HRP (horse radish peroxidase), binding at 37 deg.C for 1 hr, washing, adding TMB substrate solution for color development, reading OD with microplate reader ₄₅₀ . Positive control wells (enrichment library for each round), negative control (phage M13KO 7) and blank wells (PBS) were set. And determining that the OD value of the hole to be detected is more than 5 times of that of the negative control, and determining that the hole to be detected is positive. And (4) carrying out clone bacterium preservation, plasmid extraction and gene sequencing corresponding to the positive hole. The DNA sequence obtained by sequencing was translated into an amino acid sequence and then aligned to remove the repetitive sequence or the similar sequence differing by 1 amino acid in CDR3, to obtain unique sequences corresponding to clone numbers 1D7, 3B9 and 3G6 (the amino acid sequences of the CDR regions and the amino acid sequences of the variable regions are shown in Table 1 below, wherein the CDR regions were divided by the Kabat method)

TABLE 1 CDR region sequences and variable region sequences of single domain antibodies

Detection based on Single-Domain antibody periplasmic Chamber crude extracts (Periplasmic extracts)

S1: preparation of crude extract of single domain antibody

The unique sequence clone obtained in example 1 was subjected to the production of a crude single domain antibody extract using a bacterial expression system. The method comprises the following steps:

(1) Inoculating TG1 bacterial liquid corresponding to the unique sequence into 2ml 2 XYT culture medium containing Carb and 0.5% glucose, and culturing at 37 deg.c and 220 rpm;

(2) Detecting the optical density value of the culture solution as OD ₆₀₀ When the concentration reached 0.4, 2ml of 2 XYT medium containing IPTG (final concentration: 0.1 mM) and Carb was added and cultured overnight at 200rpm at 28 ℃;

(3) Centrifuging the bacterial liquid, collecting the thalli, resuspending the thalli by using 1ml of PBS, and then carrying out repeated freeze thawing operation for multiple times so as to release the single-domain antibody expressed in the periplasmic cavity;

(4) The cells were centrifuged (3000 rpm, 4 ℃ C., 30 minutes) and the supernatant was aspirated to obtain a crude periplasmic cavity single domain antibody extract. Meanwhile, blank TG1 bacteria without display carriers are inoculated for the same operation to prepare a blank bacteria liquid crude extract control (blank control).

S2: detection of binding Activity of Single Domain antibodies

The crude extract prepared by S1 is used for carrying out ELISA binding activity detection on IGF1R-His (product number 10164-H08H, shenzhou, yi Qiao) coated on a micropore plate, and the specific steps are as follows:

(1) Antigens were coated on 96-well plates (200 ng per well) overnight at 4 ℃. Meanwhile, setting a hole without coating antigen as a blank control;

(2) Blocking was performed at room temperature for 2 hours using 3% BSA (bovine serum albumin). During the course of the single domain antibody crude extract from the stock solution 1 dilution, each sample design three concentration gradient, respectively (1 x), 1. A crude extract of a single domain antibody targeting an unrelated target was used as a negative control (negative control) and a crude extract of a blank TG1 bacterial suspension without a display vector was used as a blank control;

(3) Washing with PBS (PBST lotion) containing 0.05% Tween-20 for 5 times, adding gradient dilution of single-domain antibody crude extract, and incubating and binding for 1 hr at room temperature under shaking;

(4) Washed 5 times with PBST, added 1;

(5) After PBST washing liquor is washed for 5 times, TMB substrate is added, the mixture is shaken at room temperature for color development for 1 minute, and 1M sulfuric acid solution is used for stopping color development;

(6) Absorbance values at 450nm were read using a microplate reader (Molecular Devices, M5 e).

The results are shown in FIG. 1, and clones 1D7, 3B9 and 3G6 have clear binding to human IGF1R.

Identification of Single Domain antibodies having the function of blocking the interaction between IGF1 and IGF1R

IGF1R-His (Pepsosers, cat # IGR-H5229) was coated in 96-well plates at 200ng per well overnight at 4 ℃. The next day, 3% BSA was added for blocking at room temperature for 2 hours, and after 5 washes with PBST, 50. Mu.l of biotin-labeled IGF1 (2. Mu.g/ml, popseud, cat # IG1-H82Q 6) and 50. Mu.L of crude single domain antibody were added. While using teportumumab at a concentration of 10. Mu.g/mL as a positive control; a crude single domain antibody extract targeting an unrelated target was used as a negative control. After 1 hour incubation at room temperature, washed 5 times with PBST, and then added 1. After 5 washes with PBST, TMB substrate was added and the development was stopped by shaking at room temperature for 1 min and finally 1M sulfuric acid solution. Absorbance values at 450nm were read using a microplate reader (Molecular Devices, M5 e). The results are shown in fig. 2, and 1D7, 3B9 and 3G6 and the positive control antibody all significantly blocked the binding of IGF1 and IGF1R.

Example 2 expression and purification of Fc fusion proteins

The pcdna3.1 recombinant vector containing the single domain antibody and the nucleic acid sequence of human IgG1 Fc (the C-terminal of VHH was directly linked to the N-terminal of human Fc hinge region) was synthesized, the human Fc region sequence used and the sequence of the constructed single domain antibody-Fc fusion protein are shown in table 2, and the constructed recombinant vector was amplified and transfected into Expi293 cells for expression of the single domain antibody-Fc fusion protein. The method comprises the following specific steps: 20 μ g of plasmid was mixed with PEI (polyethyleneimine) and incubated at room temperature for 30 minutes, after which it was added to a solution containing 1X 10 ⁶ Cell/ml in a shake flask, placed at 37 ℃ and containing 5% CO ₂ The cell culture chamber of (1) was incubated on an orbital shaker rotating at 125 rpm. On day 7 post transfection, the supernatant was harvested, purified with Protein a, and the buffer was replaced with PBS. The SDS-PAGE results of the resulting fusion proteins are shown in FIG. 3, and all have high purity.

TABLE 2 related sequences of Single Domain antibody-Fc fusion proteins

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Effect of fusion proteins on IGF1-IGF1R interactions

Recombinant human IGF1R-his (Popsex, cat. IGR-H5229) was coated in 96-well plates at 200ng per well overnight at 4 ℃. The next day, 3% BSA was added for blocking at room temperature for 2 hours, and after 5 washes with PBST, 50. Mu.L of biotin-labeled IGF1 (2. Mu.g/mL, poppsies, cat # IG1-H82Q 6) and 50. Mu.L of different concentrations of either the fusion protein test substance or the tepromumab or isotype control antibody (isotype control) were added. After 1 hour incubation at room temperature, 5 washes with PBST, followed by addition of 1. After 5 washes with PBST, TMB substrate was added and the development was stopped by shaking at room temperature for 1 min and finally 1M sulfuric acid solution. Absorbance values at 450nm were read using a microplate reader (Molecular Devices, M5 e).

As shown in FIG. 4, 1D7-Fc, 3B9-Fc, 3G6-Fc and the positive control antibody, tepitumumab, all inhibited IGF1 binding to IGF1R concentration-dependently, and 1D7-Fc and 3B9-Fc had significantly better blocking potency than the control drug, tepitumumab, with 3B9-Fc being the strongest and EC being calculated as mass concentration (ug/ml) ₅₀ The value is about 5.6 times lower than teportumumab.

Effect of fusion proteins on IGF2-IGF1R interactions

Recombinant human IGF1R-His (Pepsosex, cat IGR-H5229) was coated in 96-well plates at 200ng per well overnight at 4 ℃. The next day, 3% BSA was added for blocking for 2 hours at room temperature, and after 5 washes with PBST, 50. Mu.l of biotin-labeled IGF2 (2. Mu.g/mL, popseud, cat. IG2-H82Q 6) and 50. Mu.L of gradient-diluted fusion protein test or teprotumumab or isotype control antibody (isotype control) at different concentrations (initial final concentration 10. Mu.g/mL, 2.5 fold dilution gradient) were added. After 1 hour incubation at room temperature, washed 5 times with PBST, and then added 1. After 5 washes with PBST, TMB substrate was added and the development was stopped by shaking at room temperature for 1 min and finally 1M sulfuric acid solution. Absorbance at 450nm was read using a microplate reader (Molecular Devices, M5 e).

As shown in FIG. 5, it can be seen that 1D7-Fc, 3B9-Fc, 3G6-Fc and the positive control antibody, tepitumumab, all inhibited IGF2 binding to IGF1R concentration-dependently, consistent with the above-described results of the assay for the effect on IGF1-IGF1R interaction, as well as 1D7-Fc and 3B9-Fc had better blocking potency, with the strongest 3B9-Fc being significantly better than the control drug, tepitumumab.

Binding of fusion proteins to IGF1R of different species

The 96-well plates were coated with 5. Mu.g/mL (100. Mu.L per well) of recombinant human IGF1R-his protein (Popseud, cat. IGR-H5229), recombinant mouse IGF1R-his protein (Popseud, cat. IGR-M5223) and recombinant monkey IGF1R-his protein (Popseud, cat. IGR-C5225), respectively, overnight at 4 ℃. After blocking with 3-cent BSA for 1 hour the next day, 2. Mu.g/mL of the nanobody-Fc fusion protein sample to be tested or 4. Mu.g/mL of tepromumab, respectively, were added to the antigen-coated wells and incubated for 2 hours. Then washed 5 times with PBST and incubated for 1 hour with horseradish peroxidase conjugated anti-human Fc secondary antibody. After washing 5 times with PBST, TMB substrate was added and the color was developed for 1 minute at room temperature with shaking, and finally 1M sulfuric acid solution was added to stop the color development. Absorbance values at 450nm were read using a microplate reader (Molecular Devices, M5 e). As shown in FIG. 6, 1D7-Fc, 3B9-Fc, 3G6-Fc and teprotumumab all bound to recombinant human IGF1R-his and recombinant monkey IGF1R-his, while having no binding activity to recombinant mouse IGF 1R-his.

Example 3 Single Domain antibody humanization

Structural simulation of the 3B9 variable region sequence was performed using SWISS-MODEL, and based on the obtained structure, humanized mutation was evaluated at the camel-derived amino acid site in the sequence non-CDR region, and human germline gene V was used _3-23*01 And J _1*01 As the target sequence of the humanization mutation, a preferential mutation is located at an amino acid position on the surface of the antibody structure and not adjacent to the CDR, and a postmutation is located at a position inside the antibody structure (buried) and adjacent to the CDR. According to the principle, a plurality of humanized antibody sequences are designed: 3B9-hz1 to 3B9-hz8 (sequences as shown in Table 3)). These humanized sequences were constructed on human IgG1 Fc (sequence shown in SEQ ID NO: 13) to form a single domain antibody-Fc fusion protein. These humanized single domain antibody-Fc fusion proteins were expressed and purified by transient transfection of the Expi293 mammalian expression system.

TABLE 3 sequence of the humanized variant molecules of 3B9

Effect of humanized Single Domain antibody-Fc fusion proteins on IGF1-IGF1R interaction

Recombinant human IGF1R-his (Popsex, cat. IGR-H5229) was coated in 96-well plates at 200ng per well overnight at 4 ℃. The next day, 3% BSA was added for blocking at room temperature for 2 hours, after 5 washes with PBST, 50. Mu.l biotin-labeled IGF1 (2. Mu.g/mL, poppsies, cat # IG1-H82Q 6) and 50. Mu.l of the fusion protein assay (example 3 expressing the produced fusion protein) or teprtumumab or isotype control antibody (isotype control) at different concentrations (initial final concentration 10. Mu.g/mL, 2.5-fold dilution gradient) were added. After 1 hour incubation at room temperature, washed 5 times with PBST, and then added 1. After 5 washes with PBST, TMB substrate was added and the development was stopped by shaking at room temperature for 1 min and finally 1M sulfuric acid solution. Absorbance at 450nm was read using a microplate reader (Molecular Devices, M5 e).

As shown in FIG. 7, it can be seen that each of the humanized fusion protein molecules (3B 9-hz1-Fc to 3B9-hz 8-Fc) well retained the IGF1-IGF1R blocking potency of the parent molecule (3B 9-Fc), and the EC between each humanized molecule ₅₀ The values are closer, with the variable region sequence corresponding to the 3B9-hz8-Fc molecule having the highest level of humanization.

Affinity detection of humanized single-domain antibody-Fc fusion protein and human IGF1R

The affinity of 3B9-hz8-Fc for human IGF1R was determined by Surface Plasmon Resonance (SPR) technique using teputemumab as control and Biacore 8K (GE Healthcare) as detection device. Recombinant human IGF1R-his (Popsece, cat No. IGR-H5229) was immobilized on a CM5 chip (GE Healthcare, cat No. BR100530). Test antibodies were injected sequentially at 25 ℃ in graded dilutions at different concentrations (5, 10, 20, 40, 80 nM) with an association time of 180s and a dissociation time of 600s. The collected data were analyzed using Biacore Evaluation software. The results of the affinity constant determination of 3B9-hz8-Fc and teprotumumab for human IGF1R are shown in Table 4. It can be seen that 3B9-hz8-Fc has a higher affinity for human IGF1R than teprotumumab.

TABLE 4 binding kinetics parameters of test antibodies to human IGF1R

Ligand vs Analyte	ka(1/Ms)	kd(1/s)	Rmax(RU)	K D (M)
					3B9-hz8-Fc vs human IGF1R-his	3.97E+04	8..84E-06	62.5	2.23E-10
Teprotumumab vs human IGF1R-his	7.24E+04	6.38E-05	59.4	8.81E-10

Effect of humanized Single Domain antibody-Fc fusion proteins on IGF 1-induced tumor cell proliferation

MCF-7 tumor cells highly express IGF1R. IGF1 activates intracellular signaling pathways by binding to IGF1R, promoting cell proliferation. Testing the effect of IGF1R antibodies on IGF 1-induced proliferation of MCF-7 cells by: MCF-7 cells were seeded in 96-well cell culture plates at 20000 cells per well and 100. Mu.L of culture broth. By adding CO in a cell culture incubator (37 ℃ and 5% ₂ ) After overnight culture, the original culture medium was removed, replaced with 50. Mu.L of serum-free RPMI 1640 culture medium, starved for 6 hours, and then 25. Mu.L of test antibody compound (diluted with serum-free RPMI-1640 culture medium at an initial final concentration of 10. Mu.g/mL) was added to the wells at different concentrations and diluted in a gradient. After 1 hour of incubation, 25. Mu.L of RPMI 1640 cell culture medium containing 4% fetal bovine serum and 4ng/mL recombinant human IGF1 (Popsece, cat. IG 1-H5245) was added to each well, and the cells were placed in an incubator for further 5 days. Finally, CELL viability was determined using the CELL TITER-GLO kit according to the instructions.

As shown in FIG. 8, it can be seen that 3B9-hz8-Fc and the positive control antibody, tepitumumab, significantly inhibited MCF cell proliferation, and 3B9-hz8-Fc showed significantly better inhibitory activity than tepitumumab, with a cell residual survival rate of about 25% after treatment, compared to about 50% for tepitumumab.

Claims (9)

1. A group of IGF1R single domain antibodies, characterized in that said single domain antibodies comprise 3 unique sequences, corresponding to clone numbers 1D7, 3B9 and 3G6, each consisting of a CDR region and a variable region.

2. The group of IGF1R single domain antibodies according to claim 1, wherein the amino acid sequences of the CDR regions in said unique sequence 1D7 are shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, respectively, and the amino acid sequence of the variable region is SEQ ID No.10.

3. The group of IGF1R single domain antibodies according to claim 1, characterized in that the amino acid sequences of the CDR regions in the unique sequence 3B9 are shown as SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6, respectively, and the amino acid sequence of the variable region is SEQ ID No.11.

4. The group of IGF1R single domain antibodies of claim 1, characterized in that the amino acid sequences of the CDR regions in the unique sequence 3G6 are shown as SEQ ID No.7, SEQ ID No.8 and SEQ ID No.9, respectively, and the amino acid sequence of the variable region is SEQ ID No.12.

5. A fusion protein, characterized in that the single domain antibody-Fc fusion protein is obtained by recombining the IGF1R single domain antibody of claim 1 with a pCDNA3.1 recombinant vector having a nucleic acid sequence in which the C-terminus of VHH is directly linked to the N-terminus of human Fc hinge region.

6. The fusion protein of claim 5, wherein the human IgG1 Fc region has the sequence shown in SEQ ID No. 13; the sequence of the single-domain antibody-Fc fusion protein is shown in SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO. 16.

7. Humanized IGF1R single domain antibody, characterized in that the variable region of the single domain antibody 3B9 according to claim 1 is humanized and engineered to obtain 8 humanized antibodies 3B9-hz1, variable regions; 3B9-hz2, variable domain; 3B9-hz3, variable domain; 3B9-hz4, variable domain; 3B9-hz5, variable domain; 3B9-hz6, variable domain; 3B9-hz7, variable domain; and 3B9-hz8, variable domain; the amino acid sequences are respectively shown in SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23 and SEQ ID NO. 24.

8. Use of the IGF1R single domain antibody of claim 1, the fusion protein of claim 5 and the humanized IGF1R single domain antibody of claim 7 in the manufacture of a medicament for the treatment of a related disease induced by overexpression of IGF1R.

9. The IGF1R single domain antibody according to claim 8, the fusion protein according to claim 5 and the humanized IGF1R single domain antibody according to claim 7 for use in the treatment of related diseases induced by IGF1R overexpression, wherein the related diseases include thyroid eye disease and the like.

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