CN114984207A - anti-PD-1 nano antibody preparation - Google Patents

anti-PD-1 nano antibody preparation Download PDF

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CN114984207A
CN114984207A CN202210502742.9A CN202210502742A CN114984207A CN 114984207 A CN114984207 A CN 114984207A CN 202210502742 A CN202210502742 A CN 202210502742A CN 114984207 A CN114984207 A CN 114984207A
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CN114984207B (en
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干央央
吴幼玲
钟山
谢岩生
黄凯
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Zhejiang Teruisi Pharmaceutical Inc
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Zhejiang Teruisi Pharmaceutical Inc
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Abstract

The invention provides an anti-PD-1 nanobody preparation, which comprises an anti-PD-1 nanobody and one or more of a buffer, an osmotic pressure regulator, a viscosity regulator and a stabilizer. The anti-PD-1 nano antibody preparation provided by the invention still keeps good solubility and stability of the antibody when the antibody contains high concentration, is convenient to transport and store, and improves the validity period; and the characteristics of high-concentration nano antibody and near-human body isosmosis enable the antibody to be suitable for subcutaneous injection, and the administration of patients is convenient.

Description

anti-PD-1 nano antibody preparation
Technical Field
The invention belongs to the technical field of biological pharmacy, and particularly relates to a nano antibody preparation containing programmed death receptor 1 (PD-1).
Background
Programmed death receptor 1(PD-1) belongs to immunoglobulin superfamily CD28/B7, is I type transmembrane glycoprotein composed of 288 amino acids, and is an immunosuppressive receptor. The main ligand of PD-1 is PD-L1, which is also called B7-H1, and is a type I transmembrane glycoprotein encoded by the human CD274 gene. When PD-1 binds to PD-L1, phosphorylation of phosphatidylinositol-3-kinase, further activation of protein kinase B, activation of stimulatory T cell signaling pathways, glucose metabolism, secretion of interferon, and the like occur, which results in blocking of downstream signals of T cell activation, thereby effectively inhibiting transcription of T cells, and finally inhibiting immune function of T cells, which plays an important role in negative regulation of immune response. Therefore, the blocking of the PD-1/PD-L1 signal channel can lead the activation of T cells to be up-regulated, and activate endogenous anti-tumor immune response, thereby playing the role of treating tumors.
The stability of monoclonal antibodies is affected by changes in temperature and transport distribution during the life cycle of monoclonal antibody development, including cell metabolism in bioreactors, low pH of solutions during purification, freeze-thaw processes. Degradation pathways for monoclonal antibodies include partial unfolding, aggregation, micronization, precipitation and surface adsorption of proteins, and avoiding or reducing these physical denaturation to improve stability is an important goal of monoclonal antibody development. Monoclonal antibodies are administered by two routes, i.e., intravenous and subcutaneous. Intravenous monoclonal antibody formulations are used primarily for the treatment of diseases including tumor-targeting agents. This type of formulation requires extensive dilution. Subcutaneous injection formulations are commonly used to treat autoimmune diseases and infections and are often administered by the patient himself. To overcome the limitations of half-life and target binding capacity, protein formulations have always tended to use larger doses to reduce patient discomfort associated with large injections. However, high concentration protein formulations present a significant challenge to formulation development due to their solubility, stability and viscosity issues. Finding an appropriate prescription to improve the solubility, stability and reduce the viscosity of protein preparations becomes an important link for the development of monoclonal antibodies.
Figure BDA0003634897640000011
The monoclonal antibody injection for targeting PD-1 is developed and produced by Behcet-MeishiGuibao company, 1ml of the monoclonal antibody injection comprises 10mg of nivolumab, 5.88mg of sodium citrate dihydrate, 30mg of mannitol, 0.008mg of pentetic acid, 0.02% (w/v) of polysorbate 80 and 2.92mg of sodium chloride, and the monoclonal antibody injection is used for intravenous injection, has low concentration of the monoclonal antibody and is not suitable for subcutaneous injection.
Nanobodies (Nb), heavy chain single domain antibodies VHH (variable domain of heavy chain of heavy-chain antibodies), 2.5nm in diameter and 4nm in length, are the smallest naturally occurring fragments that can bind to an antigen. Because the structure and molecular size of the nano antibody are different from those of the traditional monoclonal antibody, in order to prepare a pharmaceutical preparation suitable for clinical application, special preparation development is carried out according to the self-structural characteristics of the nano antibody.
Disclosure of Invention
The invention aims to provide an anti-PD-1 nano antibody preparation which has high solubility and high stability and is suitable for subcutaneous injection.
In one aspect, the invention provides an anti-PD-1 nanobody formulation comprising an anti-PD-1 nanobody consisting of a VHH chain and an IgG4-Fc comprising a modified hinge region, wherein:
(i) the anti-PD-1 nanobody VHH chain comprises CDR1 shown in SEQ ID NO.6, CDR2 shown in SEQ ID NO.7, CDR3 shown in SEQ ID NO.8 and a framework region FR2 between CDR1 and CDR2 shown in SEQ ID NO. 9; and
(ii) the modified hinge region in the anti-PD-1 nano antibody comprises an amino acid sequence shown in SEQ ID NO.10, or consists of a connecting peptide and an IgG4-Fc hinge region sequence shown in SEQ ID NO.11, wherein the connecting peptide comprises an amino acid sequence shown in SEQ ID NO.12, SEQ ID NO.13 or SEQ ID NO. 14;
and, the formulation further comprises one or more of a buffer, an osmotic pressure regulator, a viscosity regulator, and a stabilizer.
The anti-PD-1 nanobody contained in the anti-PD-1 nanobody preparation provided by the present invention is disclosed in the applicant's prior chinese patent application CN202210337061.1, the entire content of which is incorporated herein for all purposes.
The amino acid sequences of the VHH chain CDR1, CDR2 and CDR3 and the amino acid sequence of the framework region FR2 in the anti-PD-1 nano antibody are respectively as follows:
SEQ ID NO.6：GSTYLRFSMG
SEQ ID NO.7：IGGDGRT
SEQ ID NO.8：AAAVLLDGSFSLLAPLVPYKYDY
SEQ ID NO.9：WFRQVPGKEREGVAA
the amino acid sequence of the modified hinge region in the anti-PD-1 nano antibody is shown as follows: SEQ ID NO. 10: ERKSSVECPPCP
Or the modified hinge region in the anti-PD-1 nano antibody consists of a connecting peptide and an IgG4-Fc hinge region sequence, wherein the IgG4-Fc hinge region sequence is shown as follows:
SEQ ID NO.11：ESKYGPPCPPCP
the linker peptide sequences are shown below:
SEQ ID NO.12：EPKIPQPQPKPQPQPQPQPKPQPKPEPE
SEQ ID NO.13：EPKIPQPQPK
SEQ ID NO.14：EPKIPQPQPKPQPKPEPE
in the case where the linker peptide is included, the linker peptide is linked to the N-terminus of the amino acid sequence shown in SEQ ID NO. 11.
Preferably, the anti-PD-1 nanobody is an anti-human PD-1 nanobody.
According to a particular embodiment of the invention, the VHH chain of said anti-PD-1 nanobody comprises the amino acid sequence shown in SEQ ID NO. 15.
SEQ ID NO.15：
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSS
Preferably, the anti-PD-1 nanobody comprises an amino acid sequence shown in SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18 or SEQ ID NO. 19.
SEQ ID NO.16：
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSERKSSVECPPCP
SEQ ID NO.17：
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKPQPQPQPQPKPQPKPEPEESKYGPPCPPCP
SEQ ID NO.18：
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKESKYGPPCPPCP
SEQ ID NO.19：
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKPQPKPEPEESKYGPPCPPCP
Further, according to a particular embodiment of the invention, said anti-PD-1 nanobody consists of a VHH chain with an IgG4-Fc comprising a modified hinge region, the full-length sequence being shown in SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 or SEQ ID No. 5.
SEQ ID NO.2：
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSERKSSVECPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
SEQ ID NO.3：
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKPQPQPQPQPKPQPKPEPEESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
SEQ ID NO.4：
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
SEQ ID NO.5：
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKPQPKPEPEESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
In the anti-PD-1 nano antibody preparation provided by the invention, the concentration of the anti-PD-1 nano antibody is 100-200mg/ml, preferably 125-175mg/ml, and more preferably 150 mg/ml.
The anti-PD-1 nano antibody preparation provided by the invention is a liquid preparation, and forms a buffer system through contained buffer solution, wherein the buffer solution is one or more selected from phosphate (sodium dihydrogen phosphate-disodium hydrogen phosphate), acetate (acetic acid-sodium acetate), citrate (citric acid-sodium citrate), Tris-HCl and histidine (histidine-histidine hydrochloric acid) buffer solution, the concentration of the buffer solution is 2-50mM, and the pH of the buffer system is 5.0-7.5, preferably 6.0-7.5, and more preferably 7.0 +/-0.2; the osmotic pressure regulator and/or the viscosity regulator is sodium chloride, and the concentration is 0-150 mM; the stabilizer is one or more selected from sucrose, trehalose and mannitol, and has a concentration of 20-200 mM.
In the buffer solution of the present invention, unless otherwise specified, phosphate refers to sodium dihydrogen phosphate-disodium hydrogen phosphate, acetate refers to acetic acid-sodium acetate, citrate refers to citric acid-sodium citrate, and histidine refers to histidine-histidine hydrochloride.
Further, the anti-PD-1 nanobody formulation further comprises a surfactant, such as a non-ionic polymer. Preferably, the surfactant is polysorbate at a concentration of 0% to 0.02%.
Preferably, the buffer is a phosphate and/or acetate buffer at a concentration of 10-20 mM. Preferably, the osmolality adjusting agent and/or viscosity adjusting agent is sodium chloride in a concentration of 0-150mM, preferably 50-100 mM. Preferably, the stabilizer is sucrose at a concentration of 30-100mM, preferably 30-65 mM. Preferably, the surfactant is polysorbate 80 at a concentration of 0% to 0.02%, more preferably 0.01% to 0.02%.
According to a specific embodiment of the present invention, in the anti-PD-1 nanobody formulation provided by the present invention, the buffer is 10mM phosphate buffer, pH 7.0; or 20mM acetate buffer, pH 5.0.
According to a specific embodiment of the present invention, the formulation comprises 150mg/ml of the anti-PD-1 nanobody and
(1) 20mM acetate buffer at pH5.0, 30mM sucrose, 100mM sodium chloride and 0.02% polysorbate 80; or
(2) 20mM acetate buffer at pH5.0, 65mM sucrose, 50mM sodium chloride and 0.01% polysorbate 80; or
(3) 20mM acetate buffer, pH5.0, 100mM sucrose and 100mM sodium chloride; or alternatively
(4) 10mM phosphate buffer pH7.0, 65mM sucrose, 50mM sodium chloride and 0.01% polysorbate 80; or alternatively
(5) 10mM phosphate buffer at pH7.0 and 30mM sucrose; or
(6) 10mM phosphate buffer pH7.0, 100mM sucrose and 100mM sodium chloride; or
(7) 10mM phosphate buffer pH7.0, 100mM sucrose and 0.02% polysorbate 80; or
(8) 10mM phosphate buffer pH7.0, 30mM sucrose, 100mM sodium chloride and 0.02% polysorbate 80.
Preferably, the anti-PD-1 nano antibody preparation provided by the invention is an injection preparation, is suitable for subcutaneous injection and intravenous injection, and is preferably a subcutaneous injection preparation or an intravenous injection preparation which is prepared by adopting sterile water for injection.
In another aspect, the invention also provides the use of the anti-PD-1 nano antibody preparation in the preparation of a medicament for treating PD-1 or PD-L1 related diseases. Preferably, the disease is a tumour (including a malignant tumour or cancer) or an infectious disease. Further preferably, the disease is lung cancer, stomach cancer, liver cancer, melanoma, cervical cancer, colorectal cancer, bladder cancer, breast cancer, leukemia, lymphoma, renal cell carcinoma, cecum cancer, pancreatic cancer, bile duct cancer, head and neck cancer, merkel cell carcinoma, ovarian cancer, nasopharyngeal cancer, glioma, esophageal cancer, bone cancer or prostate cancer. Preferably, the disease is colorectal cancer.
Accordingly, the present invention also provides a method of treating a PD-1 or PD-L1-associated disease, comprising administering to a subject in need thereof an anti-PD-1 nanobody formulation provided by the present invention. Preferably, the disease is a tumour (including a malignant tumour or cancer) or an infectious disease. Further preferably, the disease is lung cancer, stomach cancer, liver cancer, melanoma, cervical cancer, colorectal cancer, bladder cancer, breast cancer, leukemia, lymphoma, renal cell carcinoma, cecum cancer, pancreatic cancer, bile duct cancer, head and neck cancer, merkel cell carcinoma, ovarian cancer, nasopharyngeal cancer, glioma, esophageal cancer, bone cancer or prostate cancer. The subject is a vertebrate, preferably a mammal, e.g. a human, domestic and agricultural animal and a zoo, sports or pet animal, such as sheep, dog, horse, cat, cow, rat, pig, macaque. According to a particular embodiment of the invention, the subject is a human.
In yet another aspect, the invention provides other related products.
For example, a container comprising an anti-PD-1 nanobody formulation provided by the present invention. For example, the container is an injection vial, wherein the fill volume of the formulation is 1 mL/vial. Or, for example, a kit comprising a container provided herein; further, the kit further comprises instructions.
The invention develops an anti-PD-1 nano antibody preparation by taking an anti-PD-1 nano antibody as an active ingredient, wherein the preparation is clear, colorless to light yellow liquid. Experiments prove that the anti-PD-1 nano antibody has high affinity, high antigen blocking effect and good tumor inhibition effect; meanwhile, the anti-PD-1 nano antibody preparation still keeps good solubility and stability of the antibody when the antibody contains high concentration, is convenient to transport and store and improves the validity period. In addition, the anti-PD-1 nano antibody preparation has the characteristics of high concentration nano antibody and near-human body isosmosis, so that the preparation is suitable for subcutaneous injection and is convenient for patients to administer.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1: tumor volume growth trend after administration.
FIG. 2: after the experiment, the mice had heavy tumors.
Detailed Description
The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are merely illustrative of the present invention and do not limit the scope of the present invention in any way.
The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials and reagent materials used in the following examples are all commercially available products unless otherwise specified.
In the following examples, the optimized pro-sequence PR was used as a control antibody (see applicant's prior chinese patent CN107814845A), and an anti-PD-1 nanobody (see applicant's prior chinese patent application CN202210337061.1) as shown by the following amino acid sequence:
PR(SEQ ID NO.1)：
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKGLEGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
N7(SEQ ID NO.2)：
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSERKSSVECPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
wherein, VHH chain CDR1, CDR2 and CDR3 are shown in sequence in SEQ ID NOs.6-8; framework region FR2 is shown in SEQ ID NO. 9; the modified hinge region is shown in SEQ ID NO. 10; the VHH chain is shown in SEQ ID NO. 15.
N10(SEQ ID NO.3)：
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKPQPQPQPQPKPQPKPEPEESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
Wherein, VHH chain CDR1, CDR2 and CDR3 are shown in sequence in SEQ ID NOs.6-8; framework region FR2 is shown in SEQ ID NO. 9; the IgG4-Fc hinge region is shown in SEQ ID NO. 11; the connecting peptide is shown in SEQ ID NO. 12; the VHH chain is shown in SEQ ID NO. 15.
N14(SEQ ID NO.4)：
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
Wherein, the CDR1, CDR2 and CDR3 of the VHH chain are shown in sequence in SEQ ID NOs.6-8; framework region FR2 is shown in SEQ ID NO. 9; the IgG4-Fc hinge region is shown in SEQ ID NO. 11; the connecting peptide is shown in SEQ ID NO. 13; the VHH chain is shown in SEQ ID NO. 15.
N15(SEQ ID NO.5)：
QVQLQESGGGLVQPGGSLRLSCAASGSTYLRFSMGWFRQVPGKEREGVAAIGGDGRTSYADSVKGRFTISKDNSKNTLYLDMNSLRAEDTAVYYCAAAVLLDGSFSLLAPLVPYKYDYWGQGTLVTVSSEPKIPQPQPKPQPKPEPEESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
Wherein, the CDR1, CDR2 and CDR3 of the VHH chain are shown in sequence in SEQ ID NOs.6-8; framework region FR2 is shown in SEQ ID NO. 9; the IgG4-Fc hinge region is shown in SEQ ID NO. 11; the connecting peptide is shown in SEQ ID NO. 14; the VHH chain is shown in SEQ ID NO. 15.
In the following examples, the following experimental procedures were employed:
method for detecting sample
1. Appearance of the product
The color and clarity of each batch was determined visually according to the chinese pharmacopoeia 0901, 2020, color test and 0902 clarity test. Stock samples, turbidity standards and controls (WFI) were tested against a black background to determine clarity. The samples tested were compared with the colorimetric liquids (yellow line) against a white background to determine the color line.
2. Visible foreign body
Each container was gently swirled or inverted according to the chinese pharmacopoeia 0904, version 2020 to ensure no bubbles were produced and each sample was examined against background in turn. The detected visible foreign matter was recorded.
3. Protein concentration
Protein concentration was determined using a single beam diode array spectrophotometer with a 10mm quartz cuvette. The test sample was diluted to 0.5mg/mL in ultrapure water, and the absorbance was measured at 280nm and 340 nm. The concentration of the protein in the sample was determined by absorbance at 280nm and 340nm, extinction coefficient 1.50(mg/mL) -1 cm -1 And the dilution factor is calculated, and the formula is as follows:
protein concentration (mg/mL) ═ a280-a340 × 1/epsilon × D
Where A is absorbance, ε is extinction coefficient, and D is sample dilution factor.
3.SEC
Size Exclusion Chromatography (SEC) is used to determine the purity and peak area percentages of High Molecular Weight (HMW) and Low Molecular Weight (LMW) of a sample, and separation is performed based on the relationship between the pore size of the gel particles and the molecular size of the separated components.
SEC was detected by a variable wavelength uv detector using a Thermo Scientific Ultimate 3000 system. After sample dilution, the samples were placed in an autosampler for isocratic elution, and sample components were detected and integrated at a wavelength of 280 nm. The peak area percentages for high molecular weight, monomer (main peak) and low molecular weight are determined from the peak area of each component relative to the total integrated peak area.
4.IEC
Ion Exchange Chromatography (IEC) was used to determine the purity and percentage of charge variation of proteins in a sample.
IEC uses a Thermo Scientific Ultimate 3000 system with UV detection. The sample was diluted, injected and then eluted with a salt gradient, the column was kept at 35 ℃. Detection was carried out at a wavelength of 280 nm. And integrating the peaks in the chromatogram, and calculating the peak area percentages of the component peaks of the acidic peak, the main peak and the basic peak.
5.CE-SDS
a. Solution preparation: respectively preparing a dilution buffer solution, DTT, NEM, a staining gel, a destaining solution, a molecular weight standard and a denaturant;
b. sample treatment: adding ultrapure water and a reducing reagent (a sample buffer solution provided by a protein 230 kit and mixed with 1M DTT) into a sample, diluting and then denaturing; adding 120mM N-ethyl maleimide (NEM) solution and non-reducing reagent (mixing the sample solution with ultrapure water) into the sample under non-reducing condition; heating ladder (step marker) and sample under reducing and non-reducing conditions, and adding to the chip; carrying out electrophoretic separation on a sample in a micro-channel filled with a polymer solution;
c. and (3) detection: based on laser-induced fluorescence of a fluorescent dye, a sample is added to a polymer solution and non-covalently bound to protein-SDS micelles;
d. and (4) analyzing results: the bioanalyzer software automatically sized according to ladder and calculated the percentage of each separation peak in the electropherogram.
6.SVP
Insoluble particles of the sample were measured by the photoresistance method according to 2020 version pharmacopoeia of the people's republic of China, fourth 0903. Preparing four parts of each sample, and sequentially detecting; the first measurement was discarded and the remaining three were averaged and the number of particles detected above 10 μm and above 25 μm was recorded.
7. Osmotic pressure
The osmolality of the sample was determined according to the 0632 fourth part of the "pharmacopoeia of the people's republic of china" 2020 edition. The osmolarity of the finished product was determined indirectly by measuring its depression below the freezing point using an osmometer (model 210, Advanced Instruments, Norwood, MA 02062).
Example 1Antibody preparation
According to the amino acid sequence of the anti-PD-1 nanobody provided above, the corresponding nanobody is prepared by the following steps.
a. Synthesizing a gene sequence encoding a nano antibody sequence;
b. constructing the synthesized gene sequence on pcDNA3.1 vector, transforming into Escherichia coli DH5 alpha strain, shaking greatly, extracting pcDNA3.1 plasmid with Omega plasmid large extraction kit, filtering and sterilizing;
c. CHO-S cells were cultured to 5X 10 6 Per ml;
d. c, mixing the pcDNA3.1 plasmid obtained in the step b and a transfection reagent PEI in a ratio of 1:3 in a transfection medium ExpicHO TM Mixing the Expression Medium uniformly and standing for 20min, adding into the CHO-S cells of step c, and adding 6% CO at 37 deg.C 2 Culturing at 115rpm for 5 days;
e. centrifuging the culture product, and filtering with 0.2 μm filter membrane to obtain centrifugal supernatant;
f. purifying the centrifugal supernatant by adopting a ProA affinity chromatography, and washing away the foreign proteins and other impurities by using a Tris system buffer solution with the pH value of 7.0;
g. elution with 24mM acetic acid;
h. and (3) adjusting the pH of the obtained eluent back to 5.0 by using Tris Base, and filtering by using a 0.2 mu m filter membrane to obtain the antibody.
Example 2Detection of biological Activity of antibodies
2.1 antigen binding (ELISA)
And (3) detecting the binding capacity of the nano antibody to the antigen PD-1 protein by ELISA. The method comprises the following steps:
a. antigen coating: taking 100 mu L of human PD-1 protein (Acro PD1-HB2F2) with the concentration of 20ng/mL per well, incubating for 2 hours at room temperature, and washing the plate for 5 times;
b. preparing reference substances and test substances: after the reference substance and the test substance are diluted to 1000ng/mL, carrying out gradient dilution;
c. reaction: adding the sample after gradient dilution into the 96-well plate pre-coated in the step a according to 100 mu L/well, covering a plate cover, incubating at room temperature for 2 hours, and washing the plate for 5 times;
d. adding a detection antibody: transferring the diluted detection antibody to the sample plate in the step c according to 100 mu L/hole, covering a cover plate, incubating for 1 hour at room temperature, and washing the plate for 5 times;
e. adding a chromogenic substrate: adding a TMB chromogenic substrate into the sample plate, covering the cover plate, and incubating for 10-15 minutes at room temperature;
f. terminating the color development reaction: adding 2M sulfuric acid into the sample plate to terminate the color reaction;
g. reading a plate: and (4) placing the sample plate in a microplate reader, and reading the plate.
The results are shown in Table 1.
TABLE 1 antigen binding assay results for Nanobodies
Sample ID EC50(ng/ml)
PR 15.89
N7 9.98
N10 14.89
N14 15.12
N15 11.07
2.2 blocking of antigenic Activity
And detecting the blocking effect of the nano antibody on the activity of the antigen PD-1 protein. The method comprises the following steps:
a. 100mL of FBS, 10mL of NEAA, 10mL of streptomycin solution, 4mL of Hygromycin B (50mg/L) and 800 mu L of Puromycin (10mg/L) are added into 1L of DMEM/F12 basic medium to prepare CHO-PD-L1-CD3L cell complete medium; CHO-PD-L1-CD3L cells were inoculated into the medium and subcultured every 2 days to logarithmic growth phase;
100mL of FBS, 10mL of NEAA, 10mL of streptomycin solution, 8mL of Hygromycin B (50mg/L) and 400 mu L of Puromycin (10mg/L) are added into 1L of 1640 basic culture medium to prepare a Jurkat-PD-1-NFAT cell complete culture medium; Jurkat-PD-1-NFAT cells are inoculated into a culture medium, and subculture is carried out to logarithmic phase every 2 days;
b. CHO-PD-L1-CD3L cells were digested and centrifuged, and the cells were resuspended to 5X 10 with DMEM/F12 complete medium 5 Adding 100 mu L of the cells/mL into a 96-well white bottom plate per well, and incubating for 16 +/-2 h in an incubator at 37 +/-2 ℃ and (5 +/-1)% CO 2;
c. pre-diluting the test sample, the reference sample and the quality control sample to 100 mu g/mL, and performing gradient dilution by using an analysis culture medium (RPMI1640 basic culture medium + 2% FBS);
d. taking out the cell culture plate obtained in the step b, sucking supernatant liquid and discarding, and adding 50 mu L of diluted test product, reference product and quality control product into each hole;
e. Jurkat-PD-1-NFAT cells were removed and the cells were resuspended to 2X 10 with assay medium 6 Per mL; adding the Jurkat cell suspension to the culture plate of step d at 50. mu.L per well, and incubating for 6h in an incubator;
f. taking out a Bio-Glo Luciferase Assay System reagent 1h in advance, standing at room temperature, and adding 100 mu L of reagent into each hole of the cell plate; and incubating for 2-3 min in a dark place at room temperature, and reading by using an enzyme-labeling instrument.
The results are shown in Table 2.
TABLE 2 detection results of blocking effect of Nanobodies on antigen Activity
Figure BDA0003634897640000131
2.3 binding affinity (fortebio)
Detecting the binding affinity (fortebio) of the nano-antibody to the antigen PD-1 protein. The method comprises the following steps:
and (3) detecting an instrument: octet K2, pall-fortebio
Chip: protein A (manufacturer: Pall Fortebio, cat number: 18-5010)
Buffer solution: pH 7.4PBST (pH 7.4PBS, Twen200.05% v/v)
Software version: fortebio data analysis 10.0
The antibody to be detected (PR, N7, N10, N12, N13, N14 and N15) is captured through the specificity of the Protein A chip, the signal reaches 3nm, and the Protein is combined with PD-1 Protein with different concentrations diluted in a gradient manner.
Wherein, the protein of the sample to be detected is diluted by PBST buffer solution to the final concentration of 5 ug/mL. Lyophilized PD-1 protein with ddH 2 O was diluted to 250ug/mL and then diluted with PBST with a maximum concentration of 50nM, 2-fold concentration gradient, for a total of 7 concentrations.
The results are shown in Table 3.
TABLE 3 binding affinity of Nanobodies to antigens
Sample ID kon(1/Ms) koff(1/s) KD(M)
PR 2.39E+05 1.13E-03 4.72E-09
N7 2.74E+05 1.67E-04 6.10E-10
N10 2.77E+05 5.73E-04 2.07E-09
N14 2.49E+05 6.13E-04 2.46E-09
N15 3.16E+05 2.76E-04 8.73E-10
Example 3Tumor inhibition detection of nano antibody
The treatment effect of the nanobody on subcutaneous tumors was examined in a humanized mouse of C57BL/6hPD 1.
The MC38-hPD-L1 tumor cells (colon cancer cells) are transplanted subcutaneously in the C57BL/6J-hPD1 humanized mouse, and an MC38-hPD-L1 transplantation tumor model is established. The method comprises the following steps:
1. the mice are isolated and adaptively raised for one week before test treatment;
2. MC38-hPD-L1 cells were cultured at 37 deg.C with 5% CO 2 Culturing and amplifying in the incubator, collecting cells in logarithmic growth phase, suspending in PBS, adding matrigel, and adjusting cell concentration to 1 × 10 7 Individual cells/mL;
3. injecting the cell suspension into the right side of the C57BL/6J-hPD1 humanized mouse subcutaneously by a 1mL syringe, and injecting 100 μ L of each mouse;
4. the average tumor volume to be measured was about 112mm 3 When the mice were culled, they were too large, too small or had irregular tumor shapes.
Mice were divided into 5 groups by the random block method, i.e., a control group (group 1), Keytruda (group 2), PR antibody group (group 3), nanobody N7 group (group 4), nanobody N15 group (group 5), and 8 mice per group. The administration was performed according to the protocol in Table 4, with each group administered intraperitoneally at a volume of 10ml/kg for 3 weeks, 2 times per week, and 6 times total. The antibody of each group is prepared into a use concentration of 1mg/ml, and the control group adopts normal saline, and the administration is started on the grouping day.
TABLE 4 dosing regimen
Figure BDA0003634897640000151
Tumor size was measured 2 times a week, body weight of the mice was weighed, and the life status of the mice was observed and abnormal conditions were recorded. The following calculation formula is adopted:
1. tumor volume (Tumor volume, TV)
TV＝1/2×a×b 2
Wherein: a represents the tumor major axis; b represents the tumor minor axis.
2. Relative Tumor Volume (RTV)
RTV＝V t /V initial ×100(％)
Wherein, V initial When administered in groups (i.e. d) initial ) Measurement of the resulting tumor volume, V t Tumor volume at each measurement.
3. Relative tumor proliferation rate T/C (%)
T/C(％)＝(T RTV /C RTV )×100％
Wherein, T RTV Relative tumor volume, C, in the treatment groups RTV Relative tumor volumes for the solvent set are indicated.
4. Tumor volume tumor inhibition rate (TGI)
TGI＝[1-(TV t -TV initial )/(CV t -CV initial )]×100％
Wherein, TV t Represents the tumor volume at each measurement of the treatment group; TV (television) initial Represents the tumor volume of the treatment group when administered in groups; CV of t Represents the tumor volume at each measurement of the control group; CV of initial The tumor volume of the control group at the time of group administration is shown.
Results are expressed as Mean and standard error (Mean ± SEM), and differences in tumor volume between control and administered groups were analyzed using T-test, with p <0.05 indicating statistical differences.
First, the change in tumor volume in mice is shown in table 5 and fig. 1.
TABLE 5 mouse tumor volume (mm) 3 )
Figure BDA0003634897640000161
Note: data are expressed as Mean + -SEM
"-": animal euthanasia
On day 14 after the start of the administration, the mean tumor volume of the control group was 4057.43. + -. 288.73mm 3 . Group 2, group 3, group 4 and group5 the tumor volume is 884.86 +/-194.57 mm respectively 3 、1584.55±186.82mm 3 、672.45±101.01mm 3 815.51 + -131.88 mm 3 The tumor inhibition rates were 80.42%, 62.69%, 85.79% and 82.16%, respectively. Group 2, group 3, group 4 and group 5 showed significant differences (P) in tumor volume compared to the control group<0.01)。
Tumor growth inhibition of the antibodies was calculated for female humanized mice engrafted with hPD-L1 tumor based on tumor volume measured at day 14. The results are shown in tables 6 and 7.
TABLE 6 analysis of tumor growth inhibition rate
Figure BDA0003634897640000162
Figure BDA0003634897640000171
TABLE 7 comparison of tumor volumes between groups
Figure BDA0003634897640000172
Note: data are expressed as Mean + -SEM
Tumor volumes between the vehicle and treatment groups were compared using T-test analysis, indicating p <0.0001 and p < 0.05.
Second, the tumor weight of the mice
At the end of the experiment, the animals were euthanized, the tumor mass was removed and weighed, and the tumor weight of the mice was as shown in FIG. 2, specifically, the mean tumor weight of the control group was 7.0907 + -0.8349 g, and the mean tumor weights of the test samples Keytruda, PR, N7 and N15 were 3.0556 + -0.9384 g, 5.1787 + -0.3078 g, 2.4394 + -0.7359 g and 3.0210 + -0.8778 g, respectively.
The experimental results of tables 5 to 7 and fig. 1 and 2 show that the test substances N7, N15 and Keytruda antibody alone treatment achieved excellent antitumor effects on the control group in the hPD-L1-MC38 tumor model in addition to the test substance PR. N7, N15 and Keytruda were significantly superior to PR antibody in antitumor effect, and N7 and N15 showed comparable antitumor effect in hPD-L1-MC38 tumor model compared to Keytruda.
The following examples provide the establishment and preparation of the composition of the Nanobody N7(SEQ ID NO.3) preparation.
Example 4Buffer system screening of formulations
According to the protein purification process, the development process of the composition of the preparation of the present invention is divided into the development of buffer 1 and buffer 2, wherein buffer 1(UF/DF buffer) is used for ultrafiltration diafiltration, and buffer 2 (conditioning buffer) is used for diluting the protein after ultrafiltration diafiltration to the concentration required for the stock solution.
(1) Screening a preparation buffer system:
by integrating the self-structural characteristics of the nano antibody, phosphate, acetate, citrate, Tris-HCl or histidine are selected as a buffer system, and the concentration is 2-50 mM; the pH screening range is 5.0-7.5.
In addition, the osmotic pressure regulator and the viscosity regulator are NaCl with the concentration range of 0-150 mM; the stabilizer is selected from sucrose, trehalose or mannitol, and has a concentration range of 30-200 mM.
74 different solutions are obtained, and the physicochemical properties of the nano-antibody of the invention after being placed in the 74 solutions at a concentration of 100mg/ml for 7 days at 25 ℃/60% RH and then for 3 days at 40 ℃/75% RH are examined, wherein the physicochemical properties comprise detection of Size Exclusion Chromatography (SEC), non-reduction sodium dodecyl sulfate capillary gel electrophoresis (CE-SDS), insoluble microparticles (SVP) and the like. The test items and time points are shown in table 8 below.
TABLE 8.74 test items and time points for the formulations
Figure BDA0003634897640000181
The 74 formulations and their test results are shown in table 9 below.
TABLE 9.74 preparations and their test results
Figure BDA0003634897640000182
Figure BDA0003634897640000191
Figure BDA0003634897640000201
Figure BDA0003634897640000211
And on the 10 th day, when the SEC monomer peak area ratio is less than 97.5%, subsequent CE-SDS and SVP detection is not carried out, and when the CE-SDS main peak area ratio is less than 97%, SVP detection is not carried out. SVP standard, containing 10 μm and above microparticles: no more than 6000 granules/ml; containing particles of 25 μm or more: not more than 600 granules/ml.
According to the preset SEC, CE-SDS and SVP detection standards, 28 preparation samples meeting the preset standards are screened out, and the summary results are shown in the following table 10.
TABLE 10.28 formulations meeting the Preset criteria
Figure BDA0003634897640000212
Figure BDA0003634897640000221
The detection results in tables 9 and 10 show that phosphate, acetate, histidine and Tris-HCl buffer systems can be used for the nano antibody preparation, and the stability of the nano antibody preparation prepared by the citrate buffer system is relatively poor.
By combining the detection results, the buffer system with 10.0mM phosphate, pH7.0 and 20mM acetate and pH5.0 is relatively better; the sucrose as a stabilizer has better effect than mannitol and trehalose. According to the above results, buffer 1(UF/DF buffer) was selected to be 10.0mM phosphate, pH7.0 or 20mM acetate, pH 5.0; corresponding buffer 2(condition buffer) was selected from 10.0mM phosphate, sodium chloride, sucrose, pH7.0 or 20mM acetate, sodium chloride, sucrose, pH 5.0.
(2) Screening a preparation formula:
the formulation was further screened for formulation stability testing by prolonged standing and accelerated at 40 + -5 deg.C. According to the screening result of the preparation buffer, 10mM phosphate, pH7.0, 20mM acetate, pH5.0, 0-100mM sodium chloride, 30-100mM sucrose and 0% -0.02% polysorbate 80 are selected, and 10 groups of schemes are designed, and the specific scheme is shown in Table 11.
TABLE 11.10 formulation schedules
Figure BDA0003634897640000222
Figure BDA0003634897640000231
Two ultrafiltration diafiltration proteins were obtained: the concentration is 190.7mg/mL (UF/DF buffer is 10mM phosphoric acid pH 7.0); and a concentration of 203.8mg/mL (20 mM acetate for UF/DF buffer, pH5.0), according to the formulation protocol of Table 11, a formulation sample of the predetermined 150mg/mL nanobody was prepared.
Stability studies were performed by placing 10 formulation samples at 40 + -5 deg.C/75% RH for T0, 3 days, 7 days, and 14 days, and appearance, protein concentration, Ion Exchange Chromatography (IEC), Size Exclusion Chromatography (SEC), non-reducing sodium dodecyl sulfate capillary electrophoresis (non-reducing CE-SDS), insoluble microparticles (SVP), and osmolarity were measured, respectively. The test items and time points are shown in table 12.
TABLE 12 sample test items and time points
Figure BDA0003634897640000232
Figure BDA0003634897640000241
Among them, sample No. 7A was contaminated during the treatment, and only a part of the data was shown, and the rest of the test results are shown in tables 13 to 19.
TABLE 13 sample high temperature stability study appearance results
Figure BDA0003634897640000242
As can be seen from the results in Table 13, after 10 samples are placed at a high temperature of 40 +/-5 ℃ for 14 days in an accelerated manner, the samples except the sample No. 7A are clear and light yellow, and no turbidity or visible foreign matters are seen, which indicates that the buffer solution of the preparation can still ensure the good solubility of the nano antibody under the high concentration condition of 150mg/ml of the nano antibody.
TABLE 14 sample high temperature stability study concentration results (mg/ml)
Figure BDA0003634897640000243
As can be seen from the results in Table 14, the protein concentration of each sample was substantially stable after accelerated storage at high temperature for 14 days.
TABLE 15 sample high temperature stability study ion exchange chromatography results (%)
Figure BDA0003634897640000251
Sample No. 7A was contaminated, turbid at 14 days of sampling, and could not be detected
The IEC test results in table 15 show that the acetate buffer decreased the acidic and main peaks and increased the basic peak over time; phosphate buffer, with time the acidic peak tends to increase, the basic peak and the main peak slightly decrease. In general, phosphate buffer is superior to acetate buffer.
TABLE 16 sample high temperature stability study size exclusion chromatography results (%)
Figure BDA0003634897640000252
Sample No. 7A was contaminated, turbid at 14 days of sampling, and could not be detected
As can be seen from the SEC detection results in Table 16, after 10 samples are placed at high temperature for 14 days in an accelerated manner, the overall change range of monomers, polymers and fragments is small, and the phosphate buffer is slightly better than the acetate buffer.
TABLE 17 sample high temperature stability study non-reduced CE-SDS (monomer) results (%)
Figure BDA0003634897640000261
Unscheduled detection
The results of the non-reduced CE-SDS assay in Table 17 show that the antibody monomers of each sample were slightly reduced after 14 days of high-temperature storage, but the whole antibody monomers were relatively stable.
TABLE 18 results of sample high temperature stability study on insoluble microparticles
Figure BDA0003634897640000262
The insoluble particle detection results in Table 18 show that the number of insoluble particles larger than 25 μm and larger than 10 μm is increased, but the number is still far smaller than the number defined by the standard (SVP standard, containing 10 μm and more particles: not more than 6000/ml; containing 25 μm and more particles: not more than 600/ml).
Considering the subcutaneous injection administration mode, the preparation osmotic pressure and the human blood osmotic pressure are isotonic, which is favorable for subcutaneous injection administration, 5 phosphate preparation samples are subjected to osmotic pressure detection, and the detection results are shown in Table 19.
TABLE 19.5 osmotic pressure results for samples of phosphate formulations
Figure BDA0003634897640000271
After the sample is placed at 40 +/-5 ℃ for 14 days, according to the results of the osmolarity detection of 5 phosphate buffer solution preparation samples in the table 19, the osmolarity range of the normal human blood is 280-310mOsm/kg, and the osmolarity of the sample No. 10A is closer to the human isotonic.
(3) Nanobody concentration screening
Formulation samples 1B-8B were prepared according to the protocols provided in tables 20 and 21 using the above-described 10A formulation and OPDIVO formulation of the present invention, respectively, with 4 concentrations of nanobody of the present invention of 100mg/ml, 125mg/ml, 150mg/ml and 175 mg/ml.
TABLE 20 formulation of the 10A formulations of the present invention
Sample number Nanobodies Buffer solution pH Sucrose Sodium chloride Polysorbate 80
1B 100mg/ml 10mM phosphate 7.0 30mM 100mM 0.02％
2B 125mg/ml 10mM phosphate 7.0 30mM 100mM 0.02％
3B 150mg/ml 10mM phosphate 7.0 30mM 100mM 0.02％
4B 175mg/ml 10mM phosphate 7.0 30mM 100mM 0.02％
TABLE 21 OPDIVO formulation
Figure BDA0003634897640000281
A total of 8 samples of 1B-8B were placed at 40. + -. 5 ℃/75% RH for stability studies at T0, 7 days and 14 days for appearance, Ion Exchange Chromatography (IEC), Size Exclusion Chromatography (SEC) and insoluble microparticle (SVP) detection, respectively. The test items and time points are shown in table 22.
TABLE 22 sample test items and time points
Figure BDA0003634897640000282
TABLE 23 sample high temperature stability study appearance results
Figure BDA0003634897640000283
Figure BDA0003634897640000291
As can be seen from the detection results in Table 23, after the sample is accelerated and placed at a high temperature of 40 +/-5 ℃ for 14 days, the samples 1B-3B are light yellow and clear, the 4B is light yellow slight opalescence, no turbidity and visible foreign matters are observed, and the samples 5B-8B are obviously turbid when detected on the 7 th day, so that the preparation composition of the invention can better ensure the good solubility of the nano antibody preparation compared with the OPDIVO preparation under the high concentration condition of 175mg/ml of the nano antibody 100-.
TABLE 24 sample high temperature stability study ion exchange chromatography results (%)
Figure BDA0003634897640000292
Sample No. 5B-8B was very cloudy and undetectable on day 7
TABLE 25 sample high temperature stability study size exclusion chromatography results (%)
Figure BDA0003634897640000293
Figure BDA0003634897640000301
Sample No. 5B-8B was very cloudy and undetectable on day 7
TABLE 26 results of high temperature stability study on insoluble particles
Figure BDA0003634897640000302
Sample No. 5B-8B showed cloudiness at day 7, and was not examined
The detection results in tables 23 to 26 show that after the nano antibody preparations with different concentrations are placed at 40 +/-5 ℃ for 14 days, IEC, SEC and SVP are integrally stable and have no obvious abnormal change, while 4 nano antibody preparation samples prepared by adopting the OPDIVO preparation formula have obvious turbidity at 7 days.
The result is combined to show that the anti-PD-1 nano antibody has high affinity, high antigen blocking effect and good tumor inhibition effect; meanwhile, after the 100-175mg/ml high-concentration nano antibody preparation prepared from the preparation is placed at 40 +/-5 ℃ for 14 days, the appearance, the protein concentration, the IEC, the SEC, the non-reduced CE-SDS and the insoluble particles of the sample do not change abnormally, and all detection indexes meet the existing product quality standard. The nano antibody preparation keeps good solubility and stability under the condition of high-concentration nano antibody, so that the nano antibody preparation is convenient to transport and store, and the validity period is prolonged; in addition, the nanometer antibody preparation has the characteristics of high concentration of nanometer antibody and near-human body isosmosis, so that the nanometer antibody preparation is suitable for subcutaneous injection and is convenient for patients to administer.
Example 5Optimal formulations of the invention
5.1 formulation Specification
The specification of 1mL of each antibody is adopted, the concentration of the antibody is 150mg/mL, and the anti-PD-1 nano antibody in each bottle is 150 mg.
5.2 stock solution (filling the stock solution according to the preparation specification to obtain the finished product)
The stock solution of the injection is clear, colorless to light yellow liquid. The target concentrations were 150mg/mL of anti-PD-1 nanobody, 1.2mg/mL of disodium phosphate, 0.2mg/mL of sodium dihydrogen phosphate dihydrate, 5.8mg/mL of sodium chloride, 10.3mg/mL of sucrose, and 800.0002 mg/mL of polysorbate, and pH was 7.
5.3 auxiliary materials
No auxiliary materials are used in the production. All adjuvants used in the stock solution formulation were of pharmacopeia grade, listed in table 27. There is no incompatibility between these adjuvants.
TABLE 27 composition of the finished product
Composition (I) Theoretical concentration (mg/mL) Function of Execution criteria
Disodium hydrogen phosphate 1.2 Buffer solution China pharmacopoeia 2020 edition
Sodium dihydrogen phosphate dihydrate 0.2 Buffer solution China pharmacopoeia 2020 edition
Sodium chloride 5.8 Regulating seepageOsmotic pressure and viscosity China pharmacopoeia 2020 edition
Sucrose 10.3 Stabilizing agent China pharmacopoeia 2020 edition
Polysorbate 80 0.0002 Stabilizing agent China pharmacopoeia 2020 edition
Phosphate buffer, pH7.0
The function is as follows: maintaining the pH of the solution
Concentration: 10mM phosphate buffer in both stock and finished product
The phosphate buffer had good buffering capacity at pH 7.0. The concentration of 10mM phosphate buffer was sufficient to maintain the pH of the stock and finished product during the formulation process and storage of the ultrafiltration and diafiltration.
Sucrose
The function is as follows: stabilizer
Concentration: 30mM sucrose in both stock and finished product
Sucrose is used as a stabilizer. Sucrose at a concentration of 30mM is sufficient for long term storage of stable stock solutions and finished products.
Sodium chloride
The function is as follows: osmotic pressure regulating agent and viscosity regulating agent
Concentration: 100mM sodium chloride in both stock and finished product
Sodium chloride is used as an osmotic pressure and viscosity regulator, and the sodium chloride with the concentration of 100mM can meet the requirements of subcutaneous injection medicines on osmotic pressure and viscosity.
Polysorbate 80
The function is as follows: surfactants, preventing the formation of aggregates and insoluble particulates
Concentration: 0.02 percent of the stock solution and the finished product
Polysorbate 80 is a surfactant. Polysorbate 80 at a concentration of 0.02% is sufficient to maintain stability of the finished product and stock solutions during manufacture, transportation and storage.
The above description of the specific embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications according to the present invention without departing from the spirit of the present invention, which is defined by the scope of the appended claims.
Sequence listing
<110> Zhejiang Terui Si pharmaceutical Co., Ltd
<120> an anti-PD-1 nano antibody preparation
<130> LC22110027
<160> 19
<170> PatentIn version 3.5
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<213> Artificial sequence (artificial sequence)
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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Gly Leu Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu
130 135 140
Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
145 150 155 160
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
165 170 175
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
180 185 190
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
195 200 205
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
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Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
225 230 235 240
Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
245 250 255
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
260 265 270
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
275 280 285
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
290 295 300
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
305 310 315 320
Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
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Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
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Ser Leu Ser Leu Gly Lys
355
<210> 2
<211> 358
<212> PRT
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<220>
<223> N7
<400> 2
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Arg Lys Ser Ser Val Glu Cys Pro Pro Cys Pro Ala Pro Glu
130 135 140
Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
145 150 155 160
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
165 170 175
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
180 185 190
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
195 200 205
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
210 215 220
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
225 230 235 240
Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
245 250 255
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
260 265 270
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
275 280 285
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
290 295 300
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
305 310 315 320
Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
325 330 335
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
340 345 350
Ser Leu Ser Leu Gly Lys
355
<210> 3
<211> 386
<212> PRT
<213> Artificial sequence (artificial sequence)
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<223> N10
<400> 3
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Pro Lys Ile Pro Gln Pro Gln Pro Lys Pro Gln Pro Gln Pro
130 135 140
Gln Pro Gln Pro Lys Pro Gln Pro Lys Pro Glu Pro Glu Glu Ser Lys
145 150 155 160
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly
165 170 175
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
180 185 190
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu
195 200 205
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
210 215 220
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg
225 230 235 240
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
245 250 255
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
260 265 270
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
275 280 285
Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu
290 295 300
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
305 310 315 320
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
325 330 335
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
340 345 350
Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His
355 360 365
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu
370 375 380
Gly Lys
385
<210> 4
<211> 368
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> N14
<400> 4
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Pro Lys Ile Pro Gln Pro Gln Pro Lys Glu Ser Lys Tyr Gly
130 135 140
Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser
145 150 155 160
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
165 170 175
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro
180 185 190
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
195 200 205
Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val
210 215 220
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
225 230 235 240
Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr
245 250 255
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
260 265 270
Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
275 280 285
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
290 295 300
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
305 310 315 320
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser
325 330 335
Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
340 345 350
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
355 360 365
<210> 5
<211> 376
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> N15
<400> 5
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Pro Lys Ile Pro Gln Pro Gln Pro Lys Pro Gln Pro Lys Pro
130 135 140
Glu Pro Glu Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala
145 150 155 160
Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
165 170 175
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
180 185 190
Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val
195 200 205
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
210 215 220
Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
225 230 235 240
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
245 250 255
Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
260 265 270
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr
275 280 285
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
290 295 300
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
305 310 315 320
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
325 330 335
Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe
340 345 350
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
355 360 365
Ser Leu Ser Leu Ser Leu Gly Lys
370 375
<210> 6
<211> 10
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> VHH, CDR1
<400> 6
Gly Ser Thr Tyr Leu Arg Phe Ser Met Gly
1 5 10
<210> 7
<211> 7
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> VHH, CDR2
<400> 7
Ile Gly Gly Asp Gly Arg Thr
1 5
<210> 8
<211> 23
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> VHH, CDR3
<400> 8
Ala Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu
1 5 10 15
Val Pro Tyr Lys Tyr Asp Tyr
20
<210> 9
<211> 15
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> VHH, FR2
<400> 9
Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val Ala Ala
1 5 10 15
<210> 10
<211> 12
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> hinge
<400> 10
Glu Arg Lys Ser Ser Val Glu Cys Pro Pro Cys Pro
1 5 10
<210> 11
<211> 12
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> hinge
<400> 11
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro
1 5 10
<210> 12
<211> 28
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> linker
<400> 12
Glu Pro Lys Ile Pro Gln Pro Gln Pro Lys Pro Gln Pro Gln Pro Gln
1 5 10 15
Pro Gln Pro Lys Pro Gln Pro Lys Pro Glu Pro Glu
20 25
<210> 13
<211> 10
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> linker
<400> 13
Glu Pro Lys Ile Pro Gln Pro Gln Pro Lys
1 5 10
<210> 14
<211> 18
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> linker
<400> 14
Glu Pro Lys Ile Pro Gln Pro Gln Pro Lys Pro Gln Pro Lys Pro Glu
1 5 10 15
Pro Glu
<210> 15
<211> 129
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> VHH
<400> 15
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 16
<211> 141
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> VHH+hinge
<400> 16
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Arg Lys Ser Ser Val Glu Cys Pro Pro Cys Pro
130 135 140
<210> 17
<211> 169
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> VHH+linker+hinge
<400> 17
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Pro Lys Ile Pro Gln Pro Gln Pro Lys Pro Gln Pro Gln Pro
130 135 140
Gln Pro Gln Pro Lys Pro Gln Pro Lys Pro Glu Pro Glu Glu Ser Lys
145 150 155 160
Tyr Gly Pro Pro Cys Pro Pro Cys Pro
165
<210> 18
<211> 151
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> VHH+linker+hinge
<400> 18
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Pro Lys Ile Pro Gln Pro Gln Pro Lys Glu Ser Lys Tyr Gly
130 135 140
Pro Pro Cys Pro Pro Cys Pro
145 150
<210> 19
<211> 159
<212> PRT
<213> Artificial sequence (artificial sequence)
<220>
<223> VHH+linker+hinge
<400> 19
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Tyr Leu Arg Phe
20 25 30
Ser Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Ile Gly Gly Asp Gly Arg Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Asp Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Ala Val Leu Leu Asp Gly Ser Phe Ser Leu Leu Ala Pro Leu Val
100 105 110
Pro Tyr Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Pro Lys Ile Pro Gln Pro Gln Pro Lys Pro Gln Pro Lys Pro
130 135 140
Glu Pro Glu Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro
145 150 155

Claims (10)

1. An anti-PD-1 nanobody formulation comprising an anti-PD-1 nanobody consisting of a VHH chain and IgG4-Fc comprising a modified hinge region, wherein:
(i) the anti-PD-1 nanobody VHH chain comprises CDR1 shown in SEQ ID NO.6, CDR2 shown in SEQ ID NO.7, CDR3 shown in SEQ ID NO.8 and a framework region FR2 between CDR1 and CDR2 shown in SEQ ID NO. 9; and
(ii) the modified hinge region in the anti-PD-1 nano antibody comprises an amino acid sequence shown in SEQ ID NO.10, or consists of a connecting peptide and an IgG4-Fc hinge region sequence shown in SEQ ID NO.11, wherein the connecting peptide comprises an amino acid sequence shown in SEQ ID NO.12, SEQ ID NO.13 or SEQ ID NO. 14;
and, the formulation further comprises one or more of a buffer, an osmotic pressure regulator, a viscosity regulator, and a stabilizer.
2. The anti-PD-1 nanobody formulation according to claim 1, wherein the VHH chain of the anti-PD-1 nanobody comprises the amino acid sequence shown in SEQ ID No. 15;
preferably, the anti-PD-1 nano antibody comprises an amino acid sequence shown as SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18 or SEQ ID NO. 19;
more preferably, the anti-PD-1 nanobody comprises an amino acid sequence shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO. 5.
3. The anti-PD-1 nanobody preparation according to claim 1 or 2, characterized in that in the anti-PD-1 nanobody preparation, the concentration of the anti-PD-1 nanobody is 100-200mg/ml, preferably 125-175mg/ml, more preferably 150 mg/ml.
4. The anti-PD-1 nanobody formulation according to any one of claims 1 to 3, characterized in that it is a liquid formulation, said formulation forming a buffer system by means of the buffer comprised, said buffer being one or more selected from the group consisting of phosphate, acetate, citrate, Tris-HCl and histidine buffers, at a concentration of 2-50mM, said buffer system pH being 5.0-7.5, preferably 6.0-7.5, more preferably 7.0 ± 0.2;
preferably, the osmotic pressure regulator and/or the viscosity regulator is sodium chloride with the concentration of 0-150 mM;
preferably, the stabilizer is one or more selected from sucrose, trehalose and mannitol at a concentration of 20-200 mM.
5. The anti-PD-1 nanobody formulation according to any one of claims 1 to 4, characterized in that it further comprises a surfactant, such as a non-ionic polymer;
preferably, the surfactant is polysorbate at a concentration of 0% to 0.02%.
6. The anti-PD-1 nanobody formulation according to any of claims 1 to 5, characterized in that the buffer is a phosphate and/or acetate buffer, with a concentration of 10-20 mM;
preferably, the osmolality adjusting agent and/or viscosity adjusting agent is sodium chloride in a concentration of 0-150mM, preferably 50-100 mM;
preferably, the stabilizer is sucrose at a concentration of 30-100mM, preferably 30-65 mM;
preferably, the surfactant is polysorbate 80 at a concentration of 0% to 0.02%, more preferably 0.01% to 0.02%.
7. The anti-PD-1 nanobody formulation according to any one of claims 1 to 6, wherein said buffer is 10mM phosphate buffer, pH 7.0; or 20mM acetate buffer, pH 5.0.
8. The anti-PD-1 nanobody formulation according to any one of claims 1 to 7, characterized in that it comprises 150mg/ml of the anti-PD-1 nanobody and
(1) 20mM acetate buffer at pH5.0, 30mM sucrose, 100mM sodium chloride and 0.02% polysorbate 80; or
(2) 20mM acetate buffer at pH5.0, 65mM sucrose, 50mM sodium chloride and 0.01% polysorbate 80; or
(3) 20mM acetate buffer, pH5.0, 100mM sucrose and 100mM sodium chloride; or alternatively
(4) 10mM phosphate buffer, pH7.0, 65mM sucrose, 50mM sodium chloride and 0.01% polysorbate 80; or
(5) 10mM phosphate buffer at pH7.0 and 30mM sucrose; or
(6) 10mM phosphate buffer pH7.0, 100mM sucrose and 100mM sodium chloride; or
(7) 10mM phosphate buffer pH7.0, 100mM sucrose and 0.02% polysorbate 80; or alternatively
(8) 10mM phosphate buffer pH7.0, 30mM sucrose, 100mM sodium chloride and 0.02% polysorbate 80.
9. The anti-PD-1 nanobody formulation according to any one of claims 1 to 8, characterized in that it is an injectable formulation, preferably a subcutaneous or intravenous formulation;
preferably, the preparation is prepared by using sterile water for injection.
10. Use of the anti-PD-1 nanobody formulation of any one of claims 1 to 9 in the manufacture of a medicament for the treatment of a PD-1 or PD-L1-related disease;
preferably, the disease is a tumour (including a malignant tumour or cancer) or an infectious disease;
further preferably, the disease is lung cancer, stomach cancer, liver cancer, melanoma, cervical cancer, colorectal cancer, bladder cancer, breast cancer, leukemia, lymphoma, renal cell carcinoma, cecum cancer, pancreatic cancer, bile duct cancer, head and neck cancer, merkel cell carcinoma, ovarian cancer, nasopharyngeal cancer, glioma, esophageal cancer, bone cancer or prostate cancer.
CN202210502742.9A 2022-05-09 2022-05-09 anti-PD-1 nano antibody preparation Active CN114984207B (en)

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