WO2022012428A1 - Anticorps anti-pd-1 et préparation stabilisée de celui-ci - Google Patents

Anticorps anti-pd-1 et préparation stabilisée de celui-ci Download PDF

Info

Publication number
WO2022012428A1
WO2022012428A1 PCT/CN2021/105458 CN2021105458W WO2022012428A1 WO 2022012428 A1 WO2022012428 A1 WO 2022012428A1 CN 2021105458 W CN2021105458 W CN 2021105458W WO 2022012428 A1 WO2022012428 A1 WO 2022012428A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
antigen
binding fragment
variable region
chain variable
Prior art date
Application number
PCT/CN2021/105458
Other languages
English (en)
Chinese (zh)
Inventor
章燕珍
任杰
梅菲
汤沛霈
陈坤
谭小钉
Original Assignee
迈威(上海)生物科技股份有限公司
江苏迈威康新药研发有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 迈威(上海)生物科技股份有限公司, 江苏迈威康新药研发有限公司 filed Critical 迈威(上海)生物科技股份有限公司
Publication of WO2022012428A1 publication Critical patent/WO2022012428A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39591—Stabilisation, fragmentation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • 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.
  • PD-1 Programmed cell death protein 1
  • 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.
  • PD-L1 B7-H1
  • PD-L2 B7-DC
  • 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)).
  • the fully human antibody against human PD-1 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.
  • Anti-human PD-1 Libtayo mAb 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).
  • CSCC metastatic cutaneous squamous cell carcinoma
  • CSCC metastatic cutaneous squamous cell carcinoma
  • anti-human PD-1 monoclonal antibodies 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.
  • anti-PD-1 antibodies 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.
  • 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.
  • 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
  • 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.
  • 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 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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%.
  • 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
  • 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;
  • 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.
  • 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.
  • 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 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 are examples of variable region CDRs.
  • 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 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.
  • 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%.
  • the pharmaceutically acceptable adjuvant includes one or more selected from the group consisting of buffer solution, protective agent, and composition.
  • the concentration of the antibody or antigen-binding fragment contained is 15-40 mg/mL.
  • 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.
  • the protective agent is selected from the group consisting of sucrose, trehalose, mannitol, and sorbitol; the concentration is 3-10% (w/v).
  • the included surfactant is selected from polysorbate 20, polysorbate 80, EDTA, and arginine.
  • a fifth aspect, the present invention provides a composition, comprising:
  • composition of the present invention comprises:
  • composition of the present invention comprises:
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • the present invention has the following advantages over the prior art:
  • 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- ⁇ production; (5) no ADCC effector function.
  • 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.
  • 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°C for 6 months.
  • 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 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.
  • 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 ( ⁇ ) 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.
  • TRIzol kit Cat: 15596026, Invitrogen
  • M0253S, NEB M-MuLV reverse transcriptase
  • 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
  • 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.
  • CDR complementary determinant
  • 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.
  • 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 C ⁇ 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.
  • 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 ⁇ g/mL, packaged at 4°C were overnight and blocked with 5% BSA for 60 min at 37°C in a constant temperature incubator.
  • PD-1-hFc serial number: NP_005009.2, 21aa-167aa, Lot: 20180329
  • PD-1-hFc serial number: NP_005009.2, 21aa-167aa, Lot: 20180329
  • 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).
  • IC50 median effective inhibitory concentration
  • Example 4 Pharmacodynamics of h317 in PD-1 transgenic mouse xenograft model Evaluation of antitumor efficacy in vivo
  • mice 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 ⁇ L 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).
  • StudyDirector Tm version 3.1.399.19, suppliers Studylog System, Inc., S.San Francisco, CA, USA
  • 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.
  • the StudyDirector Tm version number 3.1.399.19, supplier Studylog System, Inc.
  • 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.
  • 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.
  • 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.
  • Dosing volume is 10 ⁇ L/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.
  • 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. .
  • 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.
  • 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.
  • 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.
  • 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
  • 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 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 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 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.
  • 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.
  • histidine 5.5-6.0 was selected for the next round of formula screening.
  • the histidine buffer system for further investigation, and also investigated the effects of different protein concentrations, buffer salt concentrations and protective agents on h317.
  • the investigation range of protein concentration is 15-60 mg/ml
  • the investigation range of buffer salt concentration is 5-20 mM
  • the investigation range of pH is 5.5-6.0.
  • 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 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.
  • 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.
  • 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.
  • 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 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.
  • 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 18 and Table 19 show the stability data of h317 samples at different pHs under 37°C using formulations B and H.
  • 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.
  • Example 8 The third round of screening of the components of the antibody stabilizing formulation
  • 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 ⁇ 10 ⁇ m: no more than 6000 particles; ⁇ 25 ⁇ m: 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne un anticorps se liant à la PD-1 humaine ou un fragment de celui-ci, et une composition contenant l'anticorps anti-PD-1 humaine ou son fragment, la composition étant de préférence une préparation pour injection. L'anticorps anti-PD-1 humaine réalise le criblage à l'aide d'une technologie d'hybridome pour obtenir une molécule d'anticorps 317 ayant une liaison par affinité élevée à la fois à une protéine recombinante de domaine extracellulaire de PD-1 humaine et à une protéine PD-1 humaine de surface cellulaire et capable de bloquer spécifiquement la liaison de récepteurs et de ligands de PD-1/PD-L1 et PD-1/PD-L2, et un anticorps humanisé h317 ayant une affinité et une spécificité inchangées est en outre obtenu au moyen d'une technologie d'humanisation. Selon l'analyse structurelle d'un cristal de composé antigène-anticorps, des mutants des régions CDR de chaîne légère et de chaîne lourde h317 sont construits, de telle sorte qu'un anticorps dérivé de série 317 est obtenu. Sur la base de l'anticorps anti-PD-1 humaine h317, une préparation stabilisée d'anticorps fournie au moyen de trois cycles de sélection de composant et d'optimisation de concentration améliore la stabilité au stockage et l'adaptabilité à la température de l'anticorps, et prolonge la durée de stockage de la préparation d'anticorps, en particulier d'une injection.
PCT/CN2021/105458 2020-07-14 2021-07-09 Anticorps anti-pd-1 et préparation stabilisée de celui-ci WO2022012428A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010676464 2020-07-14
CN202010676464.X 2020-07-14

Publications (1)

Publication Number Publication Date
WO2022012428A1 true WO2022012428A1 (fr) 2022-01-20

Family

ID=79274319

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/105458 WO2022012428A1 (fr) 2020-07-14 2021-07-09 Anticorps anti-pd-1 et préparation stabilisée de celui-ci

Country Status (2)

Country Link
CN (1) CN113929781A (fr)
WO (1) WO2022012428A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106390115A (zh) * 2015-07-29 2017-02-15 上海君实生物医药科技股份有限公司 一种人源化单克隆抗体的稳定制剂
CN111423510A (zh) * 2019-01-10 2020-07-17 迈威(上海)生物科技有限公司 重组抗人pd-1抗体及其应用
WO2020252418A2 (fr) * 2019-06-14 2020-12-17 Cugene, Inc. Nouveaux variants d'interleukines-2 pour le traitement du cancer
WO2021119429A1 (fr) * 2019-12-13 2021-06-17 Cugene Inc. Nouvelles protéines de fusion d'interleukine 15 (il-15) et utilisations de celles-ci
WO2021119516A1 (fr) * 2019-12-13 2021-06-17 Cugene Inc. Médicaments bioactivables à base de cytokine et procédés d'utilisations associés

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106390115A (zh) * 2015-07-29 2017-02-15 上海君实生物医药科技股份有限公司 一种人源化单克隆抗体的稳定制剂
CN111423510A (zh) * 2019-01-10 2020-07-17 迈威(上海)生物科技有限公司 重组抗人pd-1抗体及其应用
WO2020252418A2 (fr) * 2019-06-14 2020-12-17 Cugene, Inc. Nouveaux variants d'interleukines-2 pour le traitement du cancer
WO2021119429A1 (fr) * 2019-12-13 2021-06-17 Cugene Inc. Nouvelles protéines de fusion d'interleukine 15 (il-15) et utilisations de celles-ci
WO2021119516A1 (fr) * 2019-12-13 2021-06-17 Cugene Inc. Médicaments bioactivables à base de cytokine et procédés d'utilisations associés

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
WANG MINGZHU, WANG JUNCHAO, WANG RONGJUAN, JIAO SHASHA, WANG SHUANG, ZHANG JINCHAO, ZHANG MIN: "Identification of a monoclonal antibody that targets PD-1 in a manner requiring PD-1 Asn58 glycosylation", COMMUNICATIONS BIOLOGY, vol. 2, no. 1, 1 December 2019 (2019-12-01), pages 1 - 10, XP055887476, DOI: 10.1038/s42003-019-0642-9 *
WANG, M. , WANG, J. , WANG, R. , JIAO, S. , WANG, S. , ZHANG, J. , ZHANG, M: "Crystal structure of Fab of a PD-1 monoclonal antibody MW11-h317 in complex with PD-1", PROTEIN DATA BANK, 30 October 2019 (2019-10-30), pages 1 - 22, XP055887469, DOI: 10.2210/pdb6JJP/pdb *

Also Published As

Publication number Publication date
CN113929781A (zh) 2022-01-14

Similar Documents

Publication Publication Date Title
US11370837B2 (en) Anti-TIGIT antibody and use thereof
JP6993699B2 (ja) ヒトインターロイキン－２に対する免疫刺激性ヒト化モノクローナル抗体及びその融合タンパク質
CA2678181C (fr) Anticorps contre erbb3 et leur utilisation
JP6967515B2 (ja) Ｐｄ−１抗体
JP5752687B2 (ja) Ｅｒｂｂ３の外部ドメインに対する抗体およびその使用
JP2019047807A (ja) Ｐｄｇｆ受容体ベータ結合ポリペプチド
JP2022514693A (ja) Ｍｕｃ１８に特異的な抗体
JP2022523710A (ja) Ｃｄ４４に特異的な抗体
CN116323657B (zh) 同时靶向PD-L1和TGFβ的双功能分子及其医药用途
JP2022514786A (ja) Ｍｕｃ１８に特異的な抗体
WO2020143749A1 (fr) Anticorps anti-pd-1 humain recombinant et son application
WO2022012428A1 (fr) Anticorps anti-pd-1 et préparation stabilisée de celui-ci
JP2018505144A (ja) 抗ｃｘｃｌ１２抗体分子およびその使用
JP2024509369A (ja) 抗ｐｄ－ｌ１抗体及びその使用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21841995

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21841995

Country of ref document: EP

Kind code of ref document: A1