WO2022012428A1 - Anti-pd-1 antibody and stabilized preparation thereof - Google Patents

Abstract

The present invention relates to an antibody binding to human PD-1 or a fragment thereof, and a composition containing the anti-human PD-1 antibody or the fragment thereof, the composition preferably being an injection preparation. The anti-human PD-1 antibody performs screening by using a hybridoma technology to obtain an antibody molecule 317 having high affinity binding to both a human PD-1 extracellular region domain recombinant protein and a cell surface human PD-1 and capable of specifically blocking binding of receptors and ligands of PD-1/PD-L1 and PD-1/PD-L2, and a humanized antibody h317 having unchanged affinity and specificity is further obtained by means of a humanization technology is further obtained. According to structural analysis of an antigen-antibody compound crystal, mutants of h317 light and heavy chain CDR regions are constructed, so that a 317-series derivative antibody is obtained. On the basis of the anti-human PD-1 antibody h317, an antibody stabilized preparation provided by means of three rounds of component selection and concentration optimization improves the storage stability and temperature adaptability of the antibody, and prolongs the storage life of the antibody preparation especially an injection.

Description

Anti-PD-1 antibody and its stable preparation

This patent application claims the priority of the Chinese patent application with application number 202010676464.X filed on July 14, 2020, the entire contents of which are hereby incorporated by reference.

technical field

The present invention relates to the field of antibody drugs, and in particular, the present invention relates to an anti-PD-1 antibody, its stable preparation and pharmaceutical use.

Background technique

Programmed cell death protein 1 (PD-1), also known as CD279, is a member of the CD28 family of T cell receptors, expressed on the surface of a variety of immune cells, such as T cells, B cells, monocytes, etc. An important immune checkpoint molecule that inhibits the function of CD4+ and CD8+ T cells in the tumor microenvironment. Key ligands of PD-1 include PD-L1 (B7-H1) and PD-L2 (B7-DC), which are expressed by immune cells and can also be induced on a variety of tissues, when PD-1 on T cells When combined with PD-L1 or PD-L2, T cells receive inhibitory signals, thereby inhibiting T cell proliferation and cytokine production, which can effectively reduce the immune response involved in T cells. PD-L1 and PD-L2 are highly expressed on various human tumor cells, thus allowing tumor cells to escape the supervision of T cells (Pardoll DM.The blockade of immune checkpoints in cancer immunotherapy.Nat.Rev.Cancer12(4),252 – 264 (2012)).

At present, there are three types of anti-human PD-1 mAbs that have been marketed worldwide. The fully human antibody against human PD-1, Nivolumab, has been shown to inhibit the binding of PD-1 to PD-L1 and PD-L2, and is clinically used for the treatment of advanced melanoma, non-small cell lung cancer, renal cell carcinoma, Hodgkin's lymphoma, head and neck squamous cell carcinoma, urothelial carcinoma, colorectal carcinoma and hepatocellular carcinoma; humanized anti-PD-1 antibody pembrolizumab (Perbrolizumab) showed inhibition of PD-1 and PD- The combination of L1 and PD-L2 is clinically used for the treatment of malignant melanoma, non-small cell lung cancer, classic Hodgkin lymphoma, head and neck squamous cell carcinoma, d-mmr mutant or MSI-H malignancies (Alsaab et al. PD-1 and PD-L1 Checkpoint Signaling Inhibition for Cancer Immunotherapy: Mechanism, Combinations, and Clinical Outcome. Front. Pharmacol. 8:561). Anti-human PD-1 Libtayo mAb (Cemiplimab) can bind to PD-1 and block its interaction with PD-L1 and PD-L2, releasing PD-1 pathway-mediated suppression of immune responses, including anti-tumor immunity Response, clinically used for the treatment of patients with metastatic cutaneous squamous cell carcinoma (CSCC) or locally advanced CSCC (Markham, A. & Duggan, S. Drugs. 2018. doi: 10.1007/s40265-018-1012-5). There are more than 10 anti-human PD-1 monoclonal antibodies in clinical stage in China, with indications covering relapsed and refractory malignant lymphoma, Hodgkin's lymphoma, B-cell non-Hodgkin's lymphoma, acinar soft tissue sarcoma, Advanced or recurrent malignant tumor, esophageal, gastric or gastroesophageal junction cancer, nasopharyngeal carcinoma, head and neck squamous cell carcinoma, lung cancer, metastatic colorectal cancer, localized renal cell carcinoma, urothelial carcinoma, hepatocellular carcinoma , melanoma, advanced triple-negative breast cancer, pleural mesothelioma, advanced neuroendocrine tumor, unresectable or d-mmr-mutated or MSI-H solid tumor. Among them, Hengrui's SHR-1210, BeiGene's tislelizumab, and Cinda's IBI-308 have submitted listing applications, and Junshi's JS001 has been conditionally approved for listing on December 17, named toripalimab injection (trade name: Tuoyi). Internationally, there are three types of anti-PD-1 antibodies on the market, and many varieties are under clinical research. The patents involved include: WO2004004771, WO2006121168, WO2008156712, WO2010029435, WO2012145493, WO2015085847, US20170044260, CN104250302, CN105330740, CN105531288, etc.

There are many studies on antibodies against PD-1. At present, there are many anti-PD-1 antibodies on the market or in clinical trials. Although anti-PD-1 antibodies do have therapeutic effects on a variety of tumors, they often appear in specific clinical applications. Cases of poor efficacy and treatment failure. The reasons for the failure of PD-1 antibody therapy involve various factors such as the complexity of tumor diseases and individual differences of patients. Among them, the lack of stability of anti-PD-1 antibodies is also one of the important reasons affecting the effect of tumor therapy. As a biological macromolecule, anti-PD-1 antibody has a complex structure. During production and storage, physical changes such as aggregation, denaturation, and precipitation, and chemical changes such as isomerization, deamidation, and oxidation will occur. These changes can affect the safety and efficacy of the product. At present, the technical routes to improve the stability of anti-PD-1 antibodies mainly focus on: improvement and modification of antibody molecular structure, optimization and screening of antibody preparations. Since different anti-PD-1 antibodies have differences in molecular structure, binding epitopes, therapeutic activities, etc., it is necessary to study suitable stable formulations for the newly developed anti-PD-1 antibodies according to their specific conditions to reduce their use in administration. Loss of efficacy due to changes in physical and chemical properties before the subject.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide an anti-PD-L1 antibody molecule, obtain mouse antibodies by using hybridoma technology, and obtain candidate mouse antibodies through antibody activity analysis (ELISA (binding, blocking), affinity kinetics) ;Clone the variable region sequence of mouse antibody light and heavy chain gene to the upstream of the sequence encoding human antibody light and heavy chain constant region, express in mammalian cells, prepare chimeric antibody, and then block the binding of PD-1 and its ligand through antibody activity analysis Experiments, binding experiments with other CD28 family members, binding experiments with cell surface PD-1, and in vitro cytology experiments to determine the lead antibody; select a humanized template according to the Germline database, carry out humanized antibody sequence design, and obtain humanized The antibody was again tested by antibody activity analysis, blocking the binding of PD-1 to its ligands, binding to other CD28 family members, binding to cell surface PD-1, and in vitro cytology experiments. The internal and external tumor cell growth inhibition experiments were conducted for the final drugability evaluation, and the PD-1 humanized antibody sequence with the same affinity and specificity was obtained. Based on the structural analysis of the anti-PD-1 humanized antibody crystal complex, directional mutation of the CDR region was carried out to obtain antibodies with higher affinity.

Another technical problem to be solved by the present invention is that the anti-PD-1 antibody molecule is easily affected by the environment during production, transportation and storage, and the physicochemical properties are changed, which in turn leads to biological activities such as the interaction between PD-1 and PD-L1. Loss of action blocking activity; providing a stable preparation containing anti-PD-1 antibody, especially providing a stable anti-PD-1 antibody aqueous injection.

In view of the above technical problems, the purpose of the present invention is to provide an antibody or its functional fragment, and to provide its use based on the antibody or its functional fragment.

The technical solution of the present invention is as follows.

In one aspect, the present invention provides an antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein

The heavy chain variable region (VH) is selected from SEQ ID NO:54 or SEQ ID NO:62;

The light chain variable region (VL) is selected from SEQ ID NO:52 or SEQ ID NO:60.

Further, the antibodies or antigen-binding fragments thereof of the present invention are murine antibodies, chimeric antibodies, humanized antibodies, Fab, Fab', F(ab')2, Fv, and scFv.

The antibodies or fragments thereof provided by the present invention can be in any form such as monoclonal antibodies, single chain antibodies, single domain antibodies, diabodies, nanobodies, fully or partially humanized antibodies or chimeric antibodies; or, the antibodies or its fragments are half antibodies or antigen-binding fragments of half antibodies, such as scFv, BsFv, dsFv, (dsFv) ₂ , Fab, Fab', F(ab') ₂ or Fv; about the fragments of the antibodies provided by the present invention, preferably Typically, the fragment is any fragment of an antibody capable of specifically binding PD-1. The antibodies or antigen-binding fragments thereof of the present invention are murine antibodies, chimeric antibodies, humanized antibodies, Fab, Fab', F(ab')2, Fv, and scFv.

Alternatively, the antibody of the present invention is IgA, IgD, IgE, IgG or IgM, more preferably IgG1. Fragments of antibodies are selected from scFv, Fab, F(ab') ₂ or Fv fragments of said antibodies.

Preferably, the antibody or fragment thereof further comprises a human or murine constant region, preferably a human or murine light chain constant region (CL) and/or a heavy chain constant region (CH); more preferably, the antibody or Fragments thereof comprise a heavy chain constant region selected from the group consisting of IgG, IgA, IgM, IgD or IgE and/or a light chain constant region of the kappa or lambda type. According to a specific embodiment of the present invention, the antibody is a monoclonal antibody, preferably a murine, chimeric or humanized monoclonal antibody; more preferably, the heavy chain constant region of the monoclonal antibody is IgG1 or IgG4 Subtype.

In a second aspect, the present invention also provides an antibody or an antigen-binding fragment thereof, which uses the antibody or antigen-binding fragment thereof in the first aspect as a starting antibody, and based on the structural analysis of the complex between the starting antibody and PD-1 crystal, is obtained by CDRs are produced by directed evolution and at least partially retain the affinity of the starting antibody to PD-1, preferably higher than the affinity of the starting antibody to PD-1.

Further, in the antibody or antigen-binding fragment thereof of the present invention, the at least partial retention of the affinity of the starting antibody for PD-1 refers to retaining at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%.

Further, the antibody or antigen-binding fragment thereof of the present invention, wherein:

Compared with the light chain variable region of the starting antibody, the light chain variable region of the antibody or its antigen-binding fragment is selected from the group consisting of 24, 30, 32, 50, 55, 56, 91, 92, 93, 94, 96 An amino acid mutation exists at one or more of the positions; and/or

Compared with the heavy chain variable region of the starting antibody, the heavy chain variable region of the antibody or its antigen-binding fragment is selected from the group consisting of 31, 32, 33, 52, 53, 54, 55, 56, 57, 100, 101 , There is an amino acid mutation at one or more of the 106 positions;

The amino acid residue site numbering of the light chain variable region is determined according to SEQ ID NO:60; the amino acid residue site numbering of the heavy chain variable region is determined according to SEQ ID NO:62.

Further, the antibody of the present invention or its antigen-binding fragment, wherein:

The light chain variable region of the antibody or antigen-binding fragment thereof has one or more of the following amino acid substitutions compared with the light chain variable region of the starting antibody: K24R, E30S, V32A, V32Y, W50A, W50G, H55A, H55Q, T56S, Y91A, Y91F, S92D, S92N, R93N, R93S, Y94F, W96G and W96Y; and/or

The heavy chain variable region of the antibody or antigen-binding fragment thereof has one or more of the following amino acid substitutions compared with the heavy chain variable region of the starting antibody: S31D, Y32A, Y32N, D33S, D33Y, S52K, S52W, G53S, G53Y, G54D, G54S, G55S, S56G, Y57T, Y59A, Y59T, D100E, D100Y, S101A and Y106T.

Further, the antibodies or antigen-binding fragments thereof of the present invention are murine antibodies, chimeric antibodies, humanized antibodies, Fab, Fab', F(ab')2, Fv, and scFv.

In a third aspect, the present invention provides an antibody or antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof has the same light chain as the antibody or antigen-binding fragment thereof in any of the first or second aspects. Variable region CDRs and heavy chain variable region CDRs.

Further, the antibodies or antigen-binding fragments thereof of the present invention are murine antibodies, chimeric antibodies, humanized antibodies, Fab, Fab', F(ab')2, Fv, and scFv.

In a fourth aspect, the present invention provides a composition for blocking the interaction between PD-1 and its ligand, comprising the antibody or antigen-binding fragment described in any one of the first to third aspects, and optionally a pharmaceutical acceptable excipients.

Further, in the composition of the present invention, after a 37°C storage stability test, compared with before the test, after the composition is stored at 37°C for 21 days, SEC>97%, CEX>70%, NR-CE >95%.

Further, in the composition of the present invention, the pharmaceutically acceptable adjuvant includes one or more selected from the group consisting of buffer solution, protective agent, and composition.

Further, in the composition of the present invention, the concentration of the antibody or antigen-binding fragment contained is 15-40 mg/mL.

Further, the composition of the present invention, wherein, the buffer contained is selected from citrate buffer, succinate buffer, histidine buffer, phosphate buffer, acetate buffer, Tris hydrochloric acid buffer; the concentration is 5 -20 mmol; pH 4.0-8.0.

Further, in the composition of the present invention, the protective agent is selected from the group consisting of sucrose, trehalose, mannitol, and sorbitol; the concentration is 3-10% (w/v).

Further, in the composition of the present invention, the included surfactant is selected from polysorbate 20, polysorbate 80, EDTA, and arginine.

A fifth aspect, the present invention provides a composition, comprising:

Further, the composition of the present invention comprises:

Further, the composition of the present invention comprises:

In a sixth aspect, the present invention provides a composition for stabilizing an antibody, which omits the antibody or antigen-binding fragment component thereof on the basis of any one of the compositions in the fifth aspect.

In the seventh aspect, the present invention also provides the use of the composition of the sixth aspect in enhancing the stability of anti-PD-1 antibodies and/or preparing anti-PD-1 antibody preparations.

In the eighth aspect, the present invention also provides the antibody or the antigen-binding fragment thereof according to the first to third aspects of the present invention, or the composition of the fourth to seventh aspects of the present invention in the preparation of a medicament for preventing or treating tumors or cancer Applications.

In a ninth aspect, the present invention also provides a method for preventing or treating tumors or cancers, characterized in that an effective amount of any of the aforementioned antibodies or antigen-binding fragments thereof, or any of the aforementioned antibodies is administered to a subject in need thereof. said composition.

In the early stage of the present invention, human PD-1 extracellular domain recombinant protein (serial number: NP_005009.2, 21aa-167aa) was used, spleen cells were taken after immunizing mice, and the hybridoma technology was used to screen to obtain the extracellular domain of human PD-1. Both the domain recombinant protein and the cell surface human PD-1 have high affinity binding, and can specifically block the PD-1/PD-L1, PD-1/PD-L2 ligand receptor binding antibody molecules (named in the present invention). 317), further obtain a humanized antibody (named h317 in the present invention) with unchanged affinity and specificity through humanization technology, which binds, blocks, promotes T cell activation and in vivo anti-IgG4 antibody. The tumor efficacy was comprehensively evaluated, and a crystal complex of its Fab and PD-1-ECD was constructed, and its antigen-binding epitope was confirmed by crystal diffraction and further mutant validation. The experimental results of anti-PD-1 antibody molecules prove that the antibody of the present invention can effectively block the binding of PD-1 and PD-L1/PD-L1, and has a competitive binding relationship with Nivolumab and pembrolizumab (Keytruda); however, , through the crystal structure analysis of the antigen-antibody complex, it is found that the antigen-binding epitope of the antibody of the present invention is different from that of Nivolumab and Keytruda. The antibody molecule (see Invention Patent Application No. 201910022548.9, the entire content of which is incorporated by reference). The present invention further conducts preparation research on the basis of 317 series antibodies (including 317, h317, CDRs directed evolution derived antibodies), and finally determines a stable preparation formula through a unique three-round screening mode.

Specifically, the present invention has the following advantages over the prior art:

First, formulation studies were conducted for specific conditions of specific highly biologically active anti-PD-1 antibodies (317 series antibodies). The 317 series antibodies of the present invention (including 317, h317, CDRs directed evolution-derived antibodies) have: (1) block the binding of PD-1 to its ligand PD-L1 or PD-L2; (2) specifically bind to primates PD-1, does not cross-react with non-primate PD-1; (3) does not bind other CD28 family members other than PD-1; (4) induces IL-2 and/or in CD4+ T cells IFN-γ production; (5) no ADCC effector function.

Second, a unique three-round formulation optimization method is adopted. The traditional optimization method of linear formulation is to determine the parameters to be optimized with reference to the prior art, determine the sequence according to the influence of each parameter on the effect, start with the parameter that has the greatest influence on the effect, and sequentially select each parameter in a gradient. The disadvantage of this traditional formulation optimization method is that it relies too much on the ordering of the parameters to be optimized; other parameters do not reach the optimal state when the first parameter is determined, and once a parameter is selected, the subsequent optimization process of other parameters Normally this parameter will not be modified again. The present invention creatively adopts three rounds of parameter optimization. Each round of parameter optimization can not only verify the selection range of multiple parameters in the previous round/existing technology, but also provide a basis for further optimization of multiple parameters in the next round. All wheels perform joint selection and adjustment of multiple parameters.

Third, the parameter selection indicators are diversified. There are repeated verifications and different requirements between each round of screening. Finally, the stability of the three temperature conditions of 37 ° C, 25 ° C and 2-8 ° C is obtained. Preparation formula. Through the control of the above selection indicators, a stable preparation suitable for the 317 series antibodies of the present invention was obtained. After storage at 37°C for 21 days, SEC>97%, CEX>70%, NR-CE>95%; storage at 25°C for 3 months SEC>99.7%, CEX>71%; SEC>99.8%, CEX>86% when stored at 2-8℃ for 6 months.

Description of drawings

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, wherein:

Figure 1: Competitive inhibition of the binding of PD-1 to PD-L1 (6A) and PD-1 to PD-L2 (6B) by h317 antibody and Nivolumab by ELISA.

Figure 2: Statistics of tumor volume of h317 inhibiting the growth of subcutaneously transplanted tumors in PD-1 transgenic mice.

Figure 3: Process flow chart of three rounds of optimization of antibody formulation components.

Figure 4: Tm1 of h317 in different systems and pH.

Figure 5: Tagg of h317 in different systems and pH.

Figure 6: Variation trend of the SEC main peak of h317 in different systems and pH.

Figure 7: Variation trend of the CEX main peak of h317 in different systems and pH.

detailed description

The present invention will be described below with reference to specific examples. Those skilled in the art can understand that these examples are only for illustrating the present invention, and they do not limit the scope of the present invention in any way.

The experimental methods in the following examples are conventional methods unless otherwise specified. The medicinal raw materials, reagent materials, etc. used in the following examples are all commercially available products unless otherwise specified.

Example 1: Screening, identification and antibody sequence determination of anti-human PD-1 antibody hybridoma cell lines

Human PD-1 extracellular domain recombinant protein (serial number: NP_005009.2, 21aa-167aa) was used to immunize mice and spleen cells were obtained. The hybridoma technology was used to screen to obtain recombinant protein with human PD-1 extracellular domain. Both the protein and the cell surface human PD-1 have high affinity binding, and can specifically block the PD-1/PD-L1, PD-1/PD-L2 ligand receptor binding antibody molecule (named 317 in the present invention) . The total RNA of 317 hybridoma cells was extracted according to the instructions of TRIzol kit (Cat: 15596026, Invitrogen); the total RNA of hybridoma cells was reverse transcribed into cDNA using M-MuLV reverse transcriptase (Cat: M0253S, NEB); Antibody light chain variable region IgVL (κ) and heavy chain variable region VH sequences were amplified using degenerate primers and Phusion kit (Cat: E0553L, NEB); using gel recovery kit (Cat: AP-GX-250, Axygen) to purify the PCR amplification product; according to the instructions of the T vector cloning kit (Cat: ZC205, Zhuangmeng Biotechnology), the amplified PCR product was connected to the T vector and transformed into E. coli competent cells. After strain amplification and plasmid extraction, the Monoclonal antibody variable region sequences were obtained by DNA sequencing. The sequencing results show that the nucleotide sequence of the variable region DNA of the light chain of the 317 antibody is shown in SEQ ID NO: 53, and the amino acid sequence of the variable region of the light chain of the 317 antibody is deduced from the DNA sequence and shown in SEQ ID NO: 52; the heavy chain of the 317 antibody can be The nucleotide sequence of the variable region DNA is shown in SEQ ID NO: 55, and the amino acid sequence of the variable region of the 317 antibody heavy chain is deduced from the DNA sequence and shown in SEQ ID NO: 54.

Example 2: Humanization of anti-human PD-1 monoclonal antibody and construction of stable cell line

Firstly, the sequence of the heavy chain of the murine antibody was comprehensively analyzed to determine the complementary determinant (CDR) region of the antibody binding to the antigen and the framework region (framework) supporting the conserved three-dimensional conformation of the antibody. Then, according to the homology alignment results, the most similar human antibody template was found in the human antibody germline library (http://www2.mrc-lmb.cam.ac.uk/vbase/alignments2.php#VHEX), and VH3 ( 3-21) as the basic template, combined with the full sequence blast results, consider the rearranged (rearranged) antibody in the specific FR region site amino acid frequency (A49), carry out CDR transplantation, consider that the FR3 region (S98) is close to the CDR3 region, no replacement is required , according to the CDR3 sequence (Pdssgvay), JH4 (wgqgtlvtvss) was selected as the J region sequence, which realized the high degree of humanization of H1 in the framework region. According to the homology alignment results, look for the most similar human antibody template in the human antibody germline library (http://www2.mrc-lmb.cam.ac.uk/vbase/alignments2.php#VHEX), select VK II ( A19) as the basic template, combined with the full sequence blast results, considering the frequency of the amino acid occurrence of the rearranged antibody in the specific FR region (R45), CDR transplantation, according to the CDR3 sequence (Qqysrypw) situation, select JK1 (fgqgtkveik) as the JK region sequence, Full humanization of the light chain framework region is achieved.

The 317 antibody murine sequence was fully humanized and fully synthesized. The humanized 317 antibody was named h317, and the humanized h317-VH1 was cloned into the eukaryotic expression vector pKN034 by enzyme digestion. The upstream of the gene encoding the heavy chain constant region of human IgG4, the humanized h317-VL2 was cloned into the upstream of the encoding gene of the human light chain Cκ of the eukaryotic expression vector pKN019, and the humanized 317 light and heavy chains were constructed. Expression vector, h317-H and L were cloned into pKN002 eukaryotic stable high expression vector by enzyme digestion, after linearization with Pvu I, the h317 gene was integrated into CHO cell DNA using Nucleofector 2b electroporator (300452, Lonza). In , the final stable cell line with high expression of h317 was obtained through MSX (Cat: M5379-500MG, Sigma) pressure screening and subcloning. 317 The nucleotide sequence of the light chain variable region DNA after humanization is shown in SEQ ID NO: 61, and the amino acid sequence is shown in SEQ ID NO: 60; the nucleotide sequence of the heavy chain variable region DNA after humanization is shown in SEQ ID NO: 60 NO:63, see SEQ ID NO:62 for the amino acid sequence.

Example 3: ELISA detects the inhibitory effect of h317 on the binding of PD-1 to its ligands PD-L1 and PD-L2

ELISA to detect the inhibitory effect of h317 on the binding of PD-1 to PD-L1: human PD-1-hFc (serial number: NP_005009.2, 21aa-167aa, Lot: 20180329) was diluted to 0.5 μg/mL, packaged at 4°C were overnight and blocked with 5% BSA for 60 min at 37°C in a constant temperature incubator. Add h317 (Lot: DP201805002, Jiangsu Taikang Biopharmaceutical Co., Ltd.) or Nivolumab (Lot: AAW4553, BMS) (initial concentration of 3 μg/mL, 1.5-fold serial dilution), 37 ℃ constant temperature incubator reaction for 120min, add 1 μg/mL The PD-L1-mFc (serial number: NP_054862.1, 19aa-238aa, Lot: 20180412) was co-incubated with the antibody, and reacted in a constant temperature incubator at 37°C for 60 min. Then add 1:5000 diluted HRP-anti-mouse Fc (Cat: 115-035-071, Jackson Immuno Research), react for 45 minutes, add TmB (Cat: ME142, Beijing Taitianhe Biotechnology) substrate for color development for 15 minutes, 2M HCl Read the plate after termination. Taking 630nm as the reference wavelength, read and record the absorbance value A450nm-630nm of the well plate at the wavelength of 450nm.

The inhibitory effect of h317 on the binding of PD-1 to PD-L2 was detected by ELISA: PD-1-hFc (serial number: NP_005009.2, 21aa-167aa, Lot: 20180329) was diluted to 0.5 μg/mL and coated at 4°C Overnight, the cells were blocked with 5% BSA for 60 min in a constant temperature incubator at 37°C. Add h317 (Lot: DP201805002, Jiangsu Taikang Biopharmaceuticals) or Nivolumab (Lot: AAW4553, BMS) (initial concentration of 3 μg/mL, 1.5 times serial dilution), 37 ℃ constant temperature incubator reaction for 120min, add 2 μg/mL PD -L2-hFc-Biotin (serial number: NP_079515.2, 20aa-220aa, Lot: 2016.12.05), co-incubated with the antibody, and reacted in a constant temperature incubator at 37°C for 60 min. Then, 1:2000 diluted HRP-streptavidin (Cat: S2438, Sigma) was added to react for 30 min, TmB substrate was added for color development for 15 min, and the plate was read after 2M HCl was stopped. Taking 630nm as the reference wavelength, read and record the absorbance value A450nm-630nm of the well plate at the wavelength of 450nm.

The results showed that h317 could effectively block the binding of recombinant human PD-1 to its ligands PD-L1 and PD-L2. The competitive inhibitory effect of h317 and Nivolumab on the binding of PD-1 to its ligand PD-L1 was determined by ELISA, and the median effective inhibitory concentration (IC50) values were 1.4 nM and 1.3 nM, respectively (Fig. 1, 1A). The competitive inhibitory effect of h317 and Nivolumab on the binding of PD-1 to its ligand PD-L2 was determined by ELISA, and the median effective inhibitory concentration (IC50) values were 1.2 nM and 1.0 nM, respectively (Fig. 1, 1B). Compared with Nivolumab, h317 blocking activity was similar.

Example 4: Pharmacodynamics of h317 in PD-1 transgenic mouse xenograft model Evaluation of antitumor efficacy in vivo

A total of 65 HuGEMMPD-1 mice (6-8 weeks, Shanghai Southern Model Organism) were selected for the experiment, and MC38-hPD-L1 tumor cells (1×10 ⁶ / mouse, 1×10 ^{6 ) were subcutaneously inoculated on the right side of the test mice.} Cells were resuspended in 100 μL PBS). Tumor-bearing mice when the average tumor volume reaches approximately 60-100mm ^3, the measurement of animal body weight, measured with a caliper and tumor volume, starts administered prior to administration using StudyDirector ^Tm (version 3.1.399.19, suppliers Studylog System, Inc., S.San Francisco, CA, USA) were randomly divided into 6 groups for administration (D0). The number of animals in each group and the detailed administration route, dose and schedule are shown in Table 1. Administration, tumor measurement All processes such as weighing and weighing are carried out in a biological safety cabinet or ultra-clean workbench. In the experiment, the StudyDirector ^Tm (version number 3.1.399.19, supplier Studylog System, Inc.) software was used to collect data, including the measurement of the long and short diameters of the tumor and the weighing of the animal body weight. The raw data was measured by a balance and vernier calipers and directly imported into the software. The experiment was terminated when the mean tumor volume of the mice in the control group exceeded 2000 mm ³ or one week after the last administration. The tumor mass was collected, the tumor weight was measured, and the tumor photos were taken. On the day of the end of the experiment, the tumors were taken for FACS, with 4 animals in each group (corresponding to the animal number for blood collection), Marker: CD3, CD4, CD8, CD45, Live/Dead. At the end of the experiment, whole blood was taken for FACS on the day of the experiment, with 4 animals in each group (corresponding to the animal number of the tumor collected), Marker: CD3, CD4, CD8, CD45.

Table 1. Pharmacodynamic experimental design

Note: Dosing volume is 10 μL/g; hIgG control (Lot: 20180521), h317 (Lot: DP201805002, Jiangsu Taikang Biomedical), Nivolumab (Lot: AAW4553, BMS)

The HuGEMMPD-1 mouse model is a genetically engineered mouse. Under the genetic background of C57BL/6J, the human PD-1 protein coding region is inserted into the ATG position of mouse PD-1 to express human PD-1 and replace the mouse Expression of PD-1. MC38 is a murine intestinal cancer cell line induced in C57BL/6 mice. Through genetic engineering, the mouse PD-L1 of MC38 was knocked out, and the human PD-L1 was knocked in, and the MC38 cell line expressing human PD-L1 was obtained, that is, the MC38-hPD-L1 cell line. The inhibitory effect of h317 and its reference antibody Nivolumab on tumor growth was evaluated and compared in PD-1 transgenic mouse xenograft model. The results showed (see Figure 2, Table 2) that h317 significantly inhibited the growth of subcutaneously transplanted tumors in PD-1 transgenic mice at high and medium doses, and h317 was comparable to its reference antibody, Nivolumab, at high doses. .

Table 2. Tumor weight data and statistical analysis of subcutaneously transplanted tumors in PD-1 transgenic mice in each group

Notes: a. Data are presented as "mean ± standard error"; n=8.

b. The p value was obtained by one-way ANOVA of tumor volume, and there was a significant difference in F value (p<0.05), which was analyzed by Dunnett's T3 method.

Example 5: Directed mutation of h317 light and heavy chain CDR regions

1. According to the structure analysis of PD-1-h317Fab crystal complex, construct h317 light and heavy chain CDR region mutants. The construction of the expression vector is carried out by the method of plasmid site-directed mutagenesis. For details, please refer to the literature [Wang Ronghao, Chen Ruichuan, Liu Runzhong. An optimized method for fast point mutagenesis. Journal of Xiamen University (Natural Science Edition), 2008, Vol 47, sup 2, 282-285]. Mutant antibody expression and purification methods are the same as above.

2. Mutant relative affinity comparison.

The affinity comparison between the mutant and the parental antibody was done using the Octet QKe system instrument of Fortebio Company. The specific method is the same as in Example 4, except that all mutant affinities are measured in a single line to evaluate relative affinities. First, the antibody to be tested with the same target value was coated, and 100 nM human PD-1 recombinant protein (serial number: NP_005009.2, 21aa-167aa) was used as the mobile phase for relative affinity determination.

The results of the affinity determination of the mutant antibody showed that the affinity of the antibody changed greatly after multiple site mutations. The mutation site information is shown in Table 3, and the partial affinity changes are shown in Table 4.

Table 3. Amino acid sequences of the CDR regions of the h317 mutant

Table 4. Statistical table of affinity changes of h317 CDR region mutants (except for the mutation sites shown, the rest of the amino acids in the variable region are the same as h317)

From the data in the table, it can be seen that the amino acid mutation at individual positions will have a greater impact on the affinity, and the affinity of some sites is reduced by more than 10 times. At the same time, the affinity of the antibody is improved after some site mutations, such as the mutation of K at position 24 of the light chain to R, the mutation of T at position 56 to S, and the mutation of S at position 92 to N, the mutation of D at position 33 of the heavy chain After the mutation of S, the 54th G to S, and the 59th Y to A, the affinity of the antibody can be improved to a certain extent, and these mutation sites can be further mutated, and their affinity remains unchanged or further improved.

Example 6 The first round of screening of antibody stabilized formulation components

1. Screening indicators and their significance.

Due to the complexity of proteins, no single analytical method can detect all possible physical, chemical and immunological changes in protein structure. Therefore, in the process of formula screening, it is necessary to combine various analytical methods such as electrophoresis, chromatography and thermal analysis to characterize and detect changes in protein structure. Table 5 lists the assays used in this study and briefly introduces their stability-indicating effects.

Table 5. Key detection indicators of h317 formulation research

2. The effects of pH and buffer salts on the key quality attributes of h317.

The pH of protein formulations is critical for product stability and biological activity. The pH selection of the formulation is mainly affected by the physicochemical properties of the main drug molecule. Antibodies, as amphiphilic macromolecules composed of amino acids, have the following characteristics: they are prone to precipitation at pH values near the isoelectric point. After testing, the isoelectric point of h317 is 7.2. Selecting the pH of the preparation solution near the isoelectric point is likely to cause precipitation; the pH above the isoelectric point is relatively alkaline, which is not suitable for the preservation of protein substances; therefore, only Appropriate pH conditions are selected below the isoelectric point. Therefore, the pH range initially investigated in this study was 4.0-8.0.

The buffer system can prevent the slight change of pH in the preparation, thereby maintaining the stability of the protein molecule, so the buffer system suitable for the h317 molecule should be selected. Commonly used buffer systems suitable for protein drugs mainly include the following: citric acid, succinic acid, histidine, phosphoric acid, acetic acid and Tris. The h317 samples were replaced with different pH solutions prepared. The prepared solution was placed at 37°C for accelerated stability experiments, and samples were taken at 0 days, 3 days, 7 days, and 14 days for Tm/Tagg (0 days), SEC, and CEX detection. The experimental results are shown in Table 6-Table 8.

Table 6 Thermal stability data of h317 in different pH and buffer salts

Table 6 and Figure 4-5 show that the Tm1 of h317 is lower when citric acid is 4.0-5.0, succinic acid is 4.0-5.0, and acetic acid is 4.0-4.5, indicating that the samples are prone to denaturation and poor thermal stability under these conditions. The Tm1 of h317 was higher in histidine and phosphate buffer system, and Tagg was also higher than other buffers in histidine 5.5-7.0 and phosphate 7.0-8.0. Therefore, from a thermostability perspective, h317 is more stable in histidine and phosphate.

Table 7. SEC investigation results of accelerated stability of h317 in different pH and buffer salts

Table 7 and Figure 6 show that the main peaks of h317 in citric acid 4.0-4.5, phosphoric acid 7.5-8.0, succinic acid 4.0 and histidine 7.0 decreased significantly, all lower than 98%, and the increase of aggregates was obvious. It shows that low pH citric acid system and succinic acid system, high pH phosphoric acid and histidine 7.0 are not conducive to the stability of h317.

Table 8 The results of the accelerated stability CEX investigation of h317 in different pH and buffer salts

Table 8 and Figure 7 show that the main peak of CEX of h317 in citric acid 4.0 and phosphoric acid 6.5-8.0 decreased significantly, all lower than 60%, indicating that the corresponding pH values under these systems are not conducive to the stability of h317. In the investigated buffer system, the decline rate of the main peak of histidine 5.0-6.0 was the lowest, and the main peak of CEX was about 65% after 14 days at 37°C.

After comprehensive analysis of thermal stability and physical index data in the first round of screening, histidine 5.5-6.0 was selected for the next round of formula screening.

Example 7 Second round screening of antibody stabilized formulation components

According to the results of the first round of screening, we selected the histidine buffer system for further investigation, and also investigated the effects of different protein concentrations, buffer salt concentrations and protective agents on h317. Among them, the investigation range of protein concentration is 15-60 mg/ml, the investigation range of buffer salt concentration is 5-20 mM, and the investigation range of pH is 5.5-6.0. At the same time, the preliminarily investigated the effects of protective agents sucrose, trehalose, mannitol and sorbitol commonly used in biological preparations, surfactants polysorbate 80 and polysorbate 20 and other additives such as arginine and EDTA on h317. The formulation design of the formulation is shown in Table 9, and the h317 samples were replaced with the prepared solutions of different formulations. The prepared solution was placed at 37°C for accelerated stability experiments, and samples were taken at 0, 3, 7, and 21 days for SEC, CEX, and NR-CE detection.

Table 9 The second round of formula screening design

1. Protein concentration screening

Table 10 and Table 11 show the stability data of h317 samples with different protein concentrations under the condition of 37°C using formulations A, B, and C.

Table 10 Results of high temperature stability investigation of h317 in different protein concentrations

Table 11 Variation of physicochemical properties of h317 in different protein concentrations

The stability change amplitude data in Table 11 shows that the higher the concentration, the lower the SEC purity and the greater the change amplitude after 21 days at 37 °C; the NR-CE main peak of the 40 mg/ml sample decreased the most; the CEX main peak decreased with no significant difference. Therefore, considering the above stability results and the expected clinical dose, the concentration of h317 can be selected to be about 25 mg/ml.

2. Screening of protective agent types

The stability of h317 samples in different types of protective agents at 37°C was studied by formulas B, J, K, and L. The results are shown in Table 12 and Table 13.

Table 12 Results of high temperature stability investigation of h317 in different protective agents

Table 13 Variation range of physical and chemical properties of h317 in different protective agents

Table 13 shows that the main peak of SEC and CEX of h317 in mannitol has a higher decline than other protectants, and the decline of the main peak of NR-CE of trehalose and sorbitol is higher than that of sucrose and mannitol; Taken together, sucrose was selected as a protective agent for h317 in subsequent formulation studies.

3. Screening of protective agent concentration

Table 14 and Table 15 show the stability data of h317 samples in different concentrations of sucrose under the condition of 37°C using formulations B, M, N and O.

Table 14 Results of high temperature stability investigation of h317 in different sucrose concentrations

Table 15 Variation range of physical and chemical properties of h317 in different sucrose concentrations

Table 15 shows that the SEC main peak of h317 in 7% sucrose has the smallest change, indicating that 7% sucrose can protect the protein to produce less aggregates. It is shown that too high sucrose concentration may affect the charge heterogeneity, the sample in 3% sucrose has the smallest change in the main peak of NR-CE, but the main peak of SEC has the largest decrease, and above 5% sucrose has no significant difference in the change of the main peak of NR-C. Therefore, follow-up formulation studies center on 7% sucrose concentration for further investigation.

4. Screening of buffer salt concentration

Table 16 and Table 17 show the stability data of h317 samples in different buffer salt concentrations under the condition of 37°C using formulations E, B, and F.

Table 16 Results of high temperature stability investigation of h317 in different buffer salt concentrations

Table 17 Prediction results of high temperature stability period of h317 in different buffer salt concentrations

The results in Table 17 show that there is no difference in the effect of different histidine concentrations on the change amplitude of the main peak of SEC; but at 5mM, the decline range of the main peak of CEX is higher than that of the 10mM and 20mM samples; at 20mM, the decline range of NR-CE is higher than that of the other two samples. group of samples. In summary, the buffer salt of the h317 sample in the subsequent formulation study was 10 mM histidine.

5. pH screening

Table 18 and Table 19 show the stability data of h317 samples at different pHs under 37°C using formulations B and H.

Table 18 Results of high temperature stability investigation of h317 in different pH

Table 19 Variation range of physical and chemical properties of h317 in different pH

The results in Table 19 show that when h317 is at pH=5.5, the decrease of the main peak of CEX and the decrease of the main peak of NR-CE are lower than those of the pH6.0 sample, and there is no difference in the change of SEC between the two. Therefore, pH=5.5 is the center point in the follow-up formulation study. Further investigation.

6. Screening of other additives

The stability of h317 samples in other additives at 37°C was studied by using formulas B, P, Q, R, S, and T. The data results are shown in Table 20 and Table 21.

Table 20 Investigation results of high temperature stability of h317 in different pH

Table 21 Variation range of physical and chemical properties of h317 in different additives

Table 21 shows that after the addition of 3% arginine, the decrease of the SEC main peak and the decrease of the NR-CE main peak of h317 is higher than that of the control sample, indicating that arginine may cause the sample to aggregate more easily; after adding polysorbate 80 and EDTA, The decrease of the main peak of CEX is significantly higher than that of the control; based on the principle of using as few types and quantities of excipients as possible while maintaining the stability of the main drug molecule, the effect of polysorbate 20 on h317 will be further investigated in the follow-up formulation study.

The second round of screening After comprehensive analysis of physicochemical index data such as SEC, CEX, NR-CE, etc., 25mg/ml protein, 10mM histidine, 7% sucrose and 0.02% polysorbate 20, pH=5.5 as the center point The next step of prescription selection is carried out, and the next round of prescription screening is carried out.

Example 8 The third round of screening of the components of the antibody stabilizing formulation

According to the screening results of the second round, we selected 25 mg/ml protein, 10 mM histidine, 7% sucrose and 0.02% polysorbate 20 as the center point, and used JMP software to conduct a definitive screening design for the h317 prescription, where the protein concentration The investigated range is 20-30 mg/ml, the sucrose concentration is 5%-9%, the pH is 5.3-5.7, and the polysorbate 20 concentration is 0.01%-0.03%. The definitive screening design is shown in Table 22.

Table 22 The third round of formula screening design

According to the prescription design of the third round of screening described in Table 22, the SEX value and CEX value of accelerated 1 month to 3 months at 25°C and stored at 2-8°C for 1 month to 6 months were investigated. The results are shown in Tables 23-28.

Table 23 25℃ accelerated one month

Table 24 25℃ accelerated for two months

Table 25 25℃ accelerated three months

Table 26 Store at 2-8℃ for one month

Table 27 Store at 2-8℃ for three months

Table 28 2-8℃ storage for six months

From the data of 3-month acceleration at 25 degrees shown in Table 23-25, it can be seen that the aggregate has no obvious growth trend, and the acid component has increased significantly. From the current investigation results of 2-8 degrees, there is no significant difference between the groups, indicating that the sample has good stability within the dosage range of the screening preparation formulation and excipients, and the currently selected formulation is appropriate.

From the six-month 2-8 degree stability results shown in Tables 26-28, there is no significant difference between the groups. Combined with the results of three rounds of prescription screening and reference to similar drugs, it is considered appropriate to select the drug prescription. Considering that the sterilization filtration of the stock solution and the filtration of the formulation process may lead to a decrease in the polysorbate 20 content, the polysorbate 20 content was selected to be 0.02%.

After three rounds of formulation optimization and selection, the pH of the final formulation of h317 was 5.5, the protein content was 25 mg/ml, and the excipients were 10 mM histidine, 7% sucrose and 0.02% polysorbate 20, with a volume of 4.3 ml per bottle. Other quality control standards include: insoluble particles ≥ 10μm: no more than 6000 particles; ≥ 25μm: no more than 600 particles, visible foreign bodies should comply with the relevant provisions of the "Chinese Pharmacopoeia" 2015 edition of the three general rules 0904 "Visible Foreign Body Inspection Method. Both binding and biological activities were in the range of 70%-140% of the working reference.

The above description of the specific embodiments of the present invention does not limit the present invention, and those skilled in the art can make various changes or deformations according to the present invention.

Claims (21)

1. An antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein

   The heavy chain variable region (VH) is selected from SEQ ID NO:54 or SEQ ID NO:62;

   The light chain variable region (VL) is selected from SEQ ID NO:52 or SEQ ID NO:60.
2. An antibody or an antigen-binding fragment thereof, which uses the antibody or antigen-binding fragment thereof of claim 1 as a starting antibody, and is produced by CDR directed evolution transformation according to the structural analysis of the starting antibody and the PD-1 antigen crystal complex, The affinity of the starting antibody for PD-1 is at least partially retained, preferably higher than the affinity of the starting antibody for PD-1.
3. The antibody or antigen-binding fragment thereof according to claim 2, at least partially retaining the affinity of the starting antibody to PD-1 means retaining at least 10%, 20%, 30%, 40% of the affinity of the starting antibody to PD-1 , 50%, 60%, 70%, 80%, 90%, 95%.
4. The antibody or antigen-binding fragment thereof of claim 2 or 3, wherein:

   Compared with the light chain variable region of the starting antibody, the light chain variable region of the antibody or its antigen-binding fragment is selected from the group consisting of 24, 30, 32, 50, 55, 56, 91, 92, 93, 94, 96 An amino acid mutation exists at one or more of the positions; and/or

   Compared with the heavy chain variable region of the starting antibody, the heavy chain variable region of the antibody or its antigen-binding fragment is selected from the group consisting of 31, 32, 33, 52, 53, 54, 55, 56, 57, 100, 101 , There is an amino acid mutation at one or more of the 106 positions;

   The amino acid residue site numbering of the light chain variable region is determined according to SEQ ID NO:60; the amino acid residue site numbering of the heavy chain variable region is determined according to SEQ ID NO:62.
5. The antibody or antigen-binding fragment thereof of claim 4, wherein:

   The light chain variable region of the antibody or antigen-binding fragment thereof has one or more of the following amino acid substitutions compared with the light chain variable region of the starting antibody: K24R, E30S, V32A, V32Y, W50A, W50G, H55A, H55Q, T56S, Y91A, Y91F, S92D, S92N, R93N, R93S, Y94F, W96G and W96Y; and/or

   The heavy chain variable region of the antibody or antigen-binding fragment thereof has one or more of the following amino acid substitutions compared with the heavy chain variable region of the starting antibody: S31D, Y32A, Y32N, D33S, D33Y, S52K, S52W, G53S, G53Y, G54D, G54S, G55S, S56G, Y57T, Y59A, Y59T, D100E, D100Y, S101A and Y106T.
6. An antibody or antigen-binding fragment thereof, said antibody or antigen-binding fragment thereof having the same light chain variable region CDRs and heavy chain variable region CDRs as the antibody or antigen-binding fragment thereof of any one of claims 1-5.
7. The antibody or antigen-binding fragment thereof of any one of claims 1-6, which is a murine antibody, a chimeric antibody, a humanized antibody, Fab, Fab', F(ab')2, Fv, or scFv.
8. A composition for blocking the interaction between PD-1 and its ligand, comprising the antibody or antigen-binding fragment of any one of claims 1-7, and optional pharmaceutically acceptable excipients.
9. The composition according to claim 8, characterized in that after a storage stability test at 37°C, compared with before the test, after the composition is stored at 37°C for 21 days, SEC>97%, CEX>70%, NR- CE>95%.
10. The composition of claim 8 or 9, wherein the pharmaceutically acceptable adjuvant comprises one or more selected from the group consisting of a buffer, a protective agent, and a composition.
11. The composition of any one of claims 8-10, wherein the antibody or antigen-binding fragment is contained at a concentration of 15-40 mg/mL.
12. The composition according to any one of claims 8-10, wherein the buffer contained is selected from the group consisting of citrate buffer, succinate buffer, histidine buffer, phosphate buffer, acetate buffer, and Tris hydrochloric acid buffer; The concentration is 5-20 mmol; the pH is 4.0-8.0.
13. The composition according to any one of claims 8-10, wherein the protective agent is selected from the group consisting of sucrose, trehalose, mannitol, and sorbitol; the concentration is 3-10% (w/v).
14. The composition according to any one of claims 8-10, wherein the included surfactant is selected from the group consisting of polysorbate 20, polysorbate 80, EDTA, and arginine.
15. A composition comprising:

    
16. The composition of claim 15, comprising:

    
17. A composition comprising:

    

    
18. A composition for stabilizing an antibody, which is based on the composition of any one of claims 8-17, omitting the antibody or antigen-binding fragment component thereof.
19. Application of the composition of claim 18 in enhancing the stability of anti-PD-1 antibody and/or preparing anti-PD-1 antibody preparation.
20. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-7 or the composition of any one of claims 8-17 in the preparation of a medicament for preventing or treating tumors or cancer.
21. A method of preventing or treating tumor or cancer, characterized in that an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-7, or any one of claims 8-17 is administered to a subject in need the composition.

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