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WO2018032793A1 - 小分子免疫激动剂偶联pd-1抗体的新型抗体及其在抗肿瘤中的应用 - Google Patents

小分子免疫激动剂偶联pd-1抗体的新型抗体及其在抗肿瘤中的应用 Download PDF

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WO2018032793A1
WO2018032793A1 PCT/CN2017/081007 CN2017081007W WO2018032793A1 WO 2018032793 A1 WO2018032793 A1 WO 2018032793A1 CN 2017081007 W CN2017081007 W CN 2017081007W WO 2018032793 A1 WO2018032793 A1 WO 2018032793A1
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antibody
small molecule
immunoagonist
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靳广毅
王竹林
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Shenzhen Kangjuzheng Pharmaceutical Technology Co Ltd
Shenzhen University
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Shenzhen Kangjuzheng Pharmaceutical Technology Co Ltd
Shenzhen University
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    • 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
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/02—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6
    • C07D473/18—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 one oxygen and one nitrogen atom, e.g. guanine
    • 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
    • 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

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  • the present invention relates to the field of immunochemistry, and more particularly to a PD-1 antibody having an inhibitory effect on the immune system and a novel type of small molecule immunoagonist to form a novel antibody, and the preparation and resistance of the novel antibody Application in tumors.
  • PD-1 protein is an immunosuppressive protein that inhibits immune balance in normal human immunity.
  • PD-1 also inhibits the normal anti-tumor immune response; therefore, PD-1 antibodies can relieve this tumor's immunosuppressive effect (Chinese Journal of Biologicals, June 2014, Volume 27, Issue 6) 856-860), this effect has produced many long-term treatment effects in clinical treatment of tumors.
  • PD-1 antibodies have been marketed in the United States, Japan and other countries (see the following website: http://www.fiercebiotech.com/biotech/anti-pd-1-cancer-star-nivolumab-wins-world- S-first-regulatory-approval;http://www.pharmatimes.com/news/japan_approves_worlds_first_pd-1_drug%2C_nivolumab_1002153).
  • the role of the PD-1 antibody is only to relieve the "brake" of the immune system, so its efficacy is significant, but it is only effective in about 20% to 30% of patients.
  • a product is needed to simultaneously improve the autoimmune ability of tumor patients to produce synergistic effects and higher therapeutic effects.
  • the technical problem to be solved by the present invention is that the above-mentioned PD-1 antibody has a low therapeutic effect ratio, A novel antibody that conjugates a PD-1 antibody to a small molecule immunoagonist and provides its antitumor application.
  • the technical solution of the present invention is to provide a novel antibody of a small molecule immunoagonist coupled with a PD-1 antibody, which is obtained by coupling a small molecule immunoagonist and a PD-1 antibody.
  • the novel antibodies include the following compounds of the general formula [I]:
  • the X 2 represents a thiocarbonyl group:
  • the small molecule immunoagonist is a compound of formula 2-1, 2-2, 2-3:
  • the X 2 represents the following group:
  • u is an integer of 0 to 12;
  • X 2 represents the following groups:
  • u is an integer from 0 to 12;
  • X 2 represents the following groups:
  • PEG is a polyethylene glycol group
  • diethylene glycol groups Triethylene glycol group is Tetraethylene glycol group is
  • the present invention provides a novel bifunctional antibody 10, 12, 14, 16, 18 which has both an immunosuppressive release function (represented by a significantly enhanced antitumor effect) and an activated immune function.
  • the invention is created by providing both an immunosuppressive release function (with a significantly improved anti-tumor) The tumor effect is representative), and at the same time, it has a novel antibody that activates an immune function and a preparation method thereof.
  • the invention is created by providing a novel antibody having the above dual functions and promoting the proliferation of active T cells and a preparation method thereof.
  • Another object of the present invention is to provide a novel antibody prepared for use in the inhibition and stimulation of immunosuppression, antiviral, tumor immunomodulation and tumor bioimmunotherapy.
  • the novel conjugated antibody of the present invention can be used for immunotherapy and immunomodulation of malignant tumors, and the administration method can be intraperitoneal injection, subcutaneous injection, intramuscular injection, and intravenous injection; or the novel antibody and immune cells of the present invention can be used (for example, dendritic cells, natural killer cells NK, lymphocytes, monocytes/macrophages, granulocytes, etc. are co-cultured, and methods for isolating immune cells in vivo are isolated.
  • novel antibodies and the corresponding small molecule immunoagonist compounds or salts thereof for preparing the same can be formulated into various therapeutic drugs, can be combined with other drugs, synergistic drugs, or made pharmaceutically acceptable.
  • a complex or combination of carriers can be used to formulate various therapeutic drugs, can be combined with other drugs, synergistic drugs, or made pharmaceutically acceptable.
  • novel bifunctional antibodies of the invention or their salts can be used in the preparation of various ratios of therapeutic agents.
  • Figure 1 shows the interleukin-6 (IL-6) eliciting activity of novel antibodies 10, 12, 14, 16, 18.
  • FIG 2 is an interleukin-12 (IL-12)-stimulating activity of novel antibodies 10, 12, 14, 16, 18.
  • IL-12 interleukin-12
  • Figure 3 is the IFN- ⁇ eliciting activity of novel antibodies 10, 12, 14, 16, 18.
  • Figure 4 is the antitumor activity of novel antibodies 10, 12, 14, 16, 18.
  • Figure 5 is a graph showing the promotion of T cell proliferation activity of novel antibodies 10, 12, 14, 16, 18.
  • Figure 6 is the IFN- ⁇ -stimulating activity of small molecule immunoagonists 19, 20, 21, 22, 20-2.
  • Figure 7 shows interleukin-6 (IL-6) eliciting activity of small molecule immunoagonists 19, 20, 21, 22.
  • IL-6 interleukin-6
  • Figure 8 is a functional feature of the PD-1 antibody.
  • Figures 9, 10 and 11 are functional features of the PD-1 antibody of the novel antibodies of the present invention.
  • the present invention provides a novel antibody which conjugates a PD-1 antibody to a small molecule immunoagonist, and has the following structural formula [I]:
  • the X 2 represents a thiocarbonyl group:
  • the small molecule immunoagonist is a compound of formula 2-1, 2-2, 2-3:
  • the X 2 represents the following group:
  • u is an integer of 0 to 12;
  • X 2 represents the following groups:
  • u is an integer from 0 to 12;
  • X 2 represents the following groups:
  • PEG is a polyethylene glycol group
  • diethylene glycol groups Triethylene glycol group is Tetraethylene glycol group is Wait.
  • a small molecule immunoagonist is a compound represented by Formula 1, 2-1, 2-2, 2-3, and 4
  • a typical representative structural formula of a novel antibody formed by using a PD-1 antibody and other antibodies as an antibody moiety is as follows :
  • n is a number between 1 and 10.
  • the small molecule of the novel antibody-conjugated precursor is a compound represented by Formula 1, 2-1, 2-2, 2-3, and 4, a typical representative of a novel antibody formed by using a PD-1 antibody as a representative antibody is exemplified.
  • the synthesis and its structural formula are as follows:
  • the antibody is selected from the group consisting of PD-1 mAb; it can also be applied to other similar immunosuppressive antibodies, such as PD-L1 antibody, CTLA-4 antibody, TIM-3 antibody, LAG3. Antibody, TIGIT antibody, etc.; PD-1 mAb includes various monoclonal antibodies (IgG1, IgG2, and IgG4) against human PD-1 protein, and the sequence of the PD-1 protein is as follows:
  • the PD-1 antibody is directed against the sequence as an epitope sequence of an antigen.
  • the preparation method, reaction conditions and feed ratio of the coupled precursor compound 15 and 17 are the same as those of the preparation of the compound 13, except that use replace;
  • the PD-1 antibody is preferably Nivolumab (BMS-936558), Pembrolizumab (MK-3475), AMP-514, AMP-224, Pidilizumab; but is also applicable to PD-1 antibodies from other sources.
  • Nivolumab According to the method of preparing 5C4, 17D8, 2D3, 4H1, 4A11, 7D3 and 5F4 in the patent WO2006121168; and the method disclosed in CN201380062005.0.
  • Pembrolizumab is in accordance with the method disclosed in the patent WO2008156712 (A1), the preparation of h409A11, and its US Pat. No. 8,354,509 and US Pat. No. 8,900,587.
  • Pidilizumab is in accordance with the methods disclosed in the patents WO2009014708 and WO2009114335.
  • the method of preparing the PD-1 antibody can also employ other disclosed methods known in the art, such as those disclosed in Chinese Patent No. CN201410838610.9; CN201310199947.5; CN201380079581.6; CN201480011008.6; CN201310258289.2.
  • PD-1 antibody can also be purchased from the market, such as the PD-1 antibody from BioXCell, USA, name: anti h PD-1 (h CD279) IgG1, content 95%; Beijing Kehao Biotechnology Co., Ltd. anti-PD-1 Fully monoclonal antibody.
  • the source of the PD-1 antibody in the novel antibody synthesis method of the present invention is not limited thereto.
  • reaction concentrations of the following PD-1 antibodies were all 1 equivalent (1 eq, relative to other reactants).
  • Preferred antibody concentration stimulation with 0.1 ⁇ M for 24 h.
  • Peripheral monocytes were taken from volunteers, and T cells (suspended cells) were isolated and cultured at 37 ° C, 5% CO 2 serum-containing medium for 7 days, and plated at 1 ⁇ 10 6 /ml/well.
  • Preferred antibody concentration stimulation with 0.1 ⁇ M for 24 h.
  • mice 6-8 week old Balb/C mice were randomly divided into 7 groups. 2.5x10 5 4T1 tumor cells were implanted subcutaneously in the back of the mouse. The PBS blank control and 10 mg/kg of PD-1 antibody were administered respectively. The control drug and the novel antibody 10, 12, 14, 16, 18 were administered at a dose of 10 mg/kg and a volume of 100 ⁇ L. Intraperitoneal injection. On the first day after tumor implantation, each group was administered on days 7, 15, 22, and 29, respectively. Mice were euthanized when the tumor reached 1500 mm 3 or greater than 15% of body weight. Tumor inhibition results are shown in Figure 4. It can be seen that the novel antibody-treated group of the present invention has a markedly enhanced antitumor effect.
  • T cells (suspended cells) were isolated and cultured at 37 ° C, 5% CO 2 serum-containing medium for 7 days, and serum-free medium was added simultaneously with the novel antibody and PD-1 of the present invention. (All concentrations were 1 ⁇ M), and CCK-8 cells staining method was detected 3 days later. It can be seen that the novel antibodies of the present invention can promote T cell proliferation by potentiating blocking PD-1 and activating innate immunity. The result is shown in Figure 5.
  • the isolated peripheral blood lymphocytes were resuspended in physiological saline at 900 g for 10 min. Wash twice.

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Abstract

一种小分子免疫激动剂偶联PD-1抗体的偶联抗体及其应用，所述偶联抗体由小分子免疫激动剂和PD-1抗体偶联反应得到，所述偶联抗体包括通式[Ⅰ]的化合物，该偶联抗体可用于恶性肿瘤的免疫治疗和免疫调节。

Description

小分子免疫激动剂偶联PD-1抗体的新型抗体及其在抗肿瘤中的应用 技术领域
本发明涉及免疫化学领域，更具体地说，涉及一种对免疫系统具有抑制作用的PD-1抗体与一类小分子免疫激动剂化合偶联形成新型抗体，以及该新型抗体的制备和在抗肿瘤中的应用。
背景技术
近些年，在免疫抗肿瘤领域的一个重要进展是应用PD-1抗体的免疫抗肿瘤作用，作为一个抗肿瘤的突破性抗体药物，它的靶点是表达在免疫细胞上(如T细胞)的PD-1蛋白。PD-1是一个免疫抑制蛋白，对正常人体的免疫过度起到抑制和免疫平衡的作用。但是在肿瘤病人体内，PD-1也对正常的抗肿瘤免疫反应产生抑制作用；因此PD-1抗体可以解除这种对肿瘤的免疫抑制作用(中国生物制品学杂志2014年6月27卷6期856-860)，这个作用在临床治疗肿瘤中产生了许多长期治疗的效果。
目前PD-1抗体已经在美国、日本等国家上市临床应用(具体可参见以下网址：http://www.fiercebiotech.com/biotech/anti-pd-1-cancer-star-nivolumab-wins-world-s-first-regulatory-approval；http://www.pharmatimes.com/news/japan_approves_worlds_first_pd-1_drug％2C_nivolumab_1002153)。
然而，PD-1抗体的作用只是解除免疫系统的抑制“刹车”作用，因此其疗效虽然显著，但是仅仅在大约20％到30％病人中有效。为了提高其疗效比例，需要一种产品能够同时提高肿瘤病人自身免疫能力，以产生协同作用和更高的治疗效果。
发明内容
本发明要解决的技术问题在于，针对上述PD-1抗体疗效比例较低的问题， 提供一种小分子免疫激动剂偶联PD-1抗体的新型抗体及其在抗肿瘤中的应用。
本发明解决上述技术问题的技术方案是，提供一种小分子免疫激动剂偶联PD-1抗体的新型抗体，所述新型抗体由小分子免疫激动剂和PD-1抗体偶联反应得到，所述新型抗体包括以下通式[I]的化合物：
Figure PCTCN2017081007-appb-000001
式[I]中：X1表示OH或SH；R1表示烷氧基或烷基胺基，X2表示链接基团；其中抗体部分与小分子免疫激动剂组成部分由小分子免疫激动剂和所述抗体共价化合链接形成，m为小分子激动剂的数目(m定义为偶联度)，为1～10的数字；抗体部分指为抗PD-1的单抗(IgG1、IgG2或IgG4)。
当所述小分子免疫激动剂为式1的化合物时：
Figure PCTCN2017081007-appb-000002
所述X2表示硫代羰基基团：
Figure PCTCN2017081007-appb-000003
当所述小分子免疫激动剂为式2-1、2-2、2-3的化合物时：
Figure PCTCN2017081007-appb-000004
所述X2表示如下基团：
Figure PCTCN2017081007-appb-000005
当所述小分子免疫激动剂为式3的化合物时：
Figure PCTCN2017081007-appb-000006
式3中，u为0～12的整数；
X2表示如下基团：
Figure PCTCN2017081007-appb-000007
其中u为0～12的整数；
当所述小分子免疫激动剂为式4的化合物时：
Figure PCTCN2017081007-appb-000008
X2表示如下基团：
Figure PCTCN2017081007-appb-000009
其中PEG为聚乙二醇基团
Figure PCTCN2017081007-appb-000010
如二聚乙二醇基团为
Figure PCTCN2017081007-appb-000011
三聚乙二醇基团为
Figure PCTCN2017081007-appb-000012
四聚乙二醇基团为
Figure PCTCN2017081007-appb-000013
本发明提供了一种新型的双功能抗体10、12、14、16、18，这些新型抗体既有免疫抑制解除功能(以显著提高的抗肿瘤效果为代表)，又同时具有激活免疫功能。
本发明的创造之处在于，提供既有免疫抑制解除功能(以显著提高的抗肿 瘤效果为代表)，又同时具有激活免疫功能的新型抗体及其制备方法。
本发明的创造之处在于，提供具有上述双功能，又能促进活性T细胞增值的新型抗体及其制备方法。
本发明的目的在于，提供用于制备上述新型抗体的偶联前体小分子化合物及合成这些偶联前体小分子的化合物或它们的盐。
本发明的另一目的在于，提供所制备的新型抗体在免疫抑制的解除和激发、抗病毒、肿瘤免疫调节和肿瘤生物免疫治疗中的应用。
本发明中的新偶联抗体可用于恶性肿瘤的免疫治疗和免疫调节，其用药方法可为腹腔注射、皮下注射、肌肉注射和静脉注射；或者可采用将本发明中的新型抗体和免疫细胞(如树突状细胞、自然杀伤细胞NK、淋巴细胞、单核/巨噬细胞、粒细胞等)共培养后，分离免疫细胞体内回输的方法。
上述新型抗体和制备它们的相应的小分子免疫激动剂化合物或它们的盐可制成适用于上述各种治疗药物、可与其他药物制成复方药物、协同药物、或制成药学上可接受的载体的复合物或结合物。
本发明的新型双功能抗体或它们的盐可用于制备各种比例的治疗药物中。
附图说明
图1是新型抗体10、12、14、16、18的白介素-6(IL-6)激发活性。
图2是新型抗体10、12、14、16、18的白介素-12(IL-12)激发活性。
图3是新型抗体10、12、14、16、18的IFN-γ激发活性。
图4是新型抗体10、12、14、16、18的抗肿瘤活性。
图5是新型抗体10、12、14、16、18的促进T细胞增值活性。
图6是小分子免疫激动剂19、20、21、22、20-2的IFN-γ激发活性。
图7是小分子免疫激动剂19、20、21、22的白介素-6(IL-6)激发活性。
图8是PD-1抗体的功能特征。
图9、10、11是本发明新型抗体的PD-1抗体功能特征。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
本发明提供了一种小分子免疫激动剂偶联PD-1抗体的新型抗体，具有如下的结构通式[I]表示：
Figure PCTCN2017081007-appb-000014
式[I]中：X1表示OH或SH；R1表示烷氧基或烷基胺基，X2表示链接基团；其中抗体与小分子免疫激动剂组成部分由小分子免疫激动剂和所述抗体共价化合链接形成，m为小分子激动剂的数目(m定义为偶联度)，为1～10的数字；抗体部分指为抗PD-1的单抗(IgG1、IgG2或IgG4)。
当所述小分子免疫激动剂为式1的化合物时：
Figure PCTCN2017081007-appb-000015
所述X2表示硫代羰基基团：
Figure PCTCN2017081007-appb-000016
当所述小分子免疫激动剂为式2-1、2-2、2-3的化合物时：
Figure PCTCN2017081007-appb-000017
Figure PCTCN2017081007-appb-000018
所述X2表示如下基团：
Figure PCTCN2017081007-appb-000019
当所述小分子免疫激动剂为式3的化合物时：
Figure PCTCN2017081007-appb-000020
式3中，u为0～12的整数；
X2表示如下基团：
Figure PCTCN2017081007-appb-000021
其中u为0～12的整数；
当所述小分子免疫激动剂为式4的化合物时：
Figure PCTCN2017081007-appb-000022
X2表示如下基团：
Figure PCTCN2017081007-appb-000023
其中PEG为聚乙二醇基团
Figure PCTCN2017081007-appb-000024
如二聚乙二醇基团为
Figure PCTCN2017081007-appb-000025
三聚乙二醇基团为
Figure PCTCN2017081007-appb-000026
四聚乙二醇基团为
Figure PCTCN2017081007-appb-000027
等。
当小分子免疫激动剂为式1、2-1、2-2、2-3、4代表的化合物时，以PD-1抗体和其它抗体为例作为抗体部分形成的新型抗体的典型代表结构式如下：
Figure PCTCN2017081007-appb-000028
Figure PCTCN2017081007-appb-000029
其中m为1到10之间的数。
当偶联新型抗体的前体小分子为式1、2-1、2-2、2-3、4代表的化合物时，以PD-1抗体为例作为代表性抗体形成的新型抗体的典型代表合成及其结构式如下：
Figure PCTCN2017081007-appb-000030
Figure PCTCN2017081007-appb-000031
Figure PCTCN2017081007-appb-000032
在上述偶联形成的新型抗体中，抗体选自PD-1单抗；也可应用于其它的类似的解除免疫抑制的抗体，如PD-L1抗体、CTLA-4抗体、TIM-3抗体、LAG3抗体、TIGIT抗体等；PD-1单抗包括各种抗人PD-1蛋白的单抗(IgG1、IgG2和IgG4)，所述PD-1蛋白的序列如下：
Figure PCTCN2017081007-appb-000033
所述PD-1抗体针对该序列为抗原的表位序列。
偶联前体化合物11合成举例：
Figure PCTCN2017081007-appb-000034
将20-1溶解于无水DMSO中，于10℃冷却加入等当量的丁二酸酐，混合物自然室温搅拌24小时。混合反应物倒与20倍体积水中，析出大量白色固体化合物SZU-101；收率88％；MS(ESI)445.27(M+1)；
将SZU-101(1eq)，NHS(1.2eq)和EDC(1.3eq)溶于无水DMF，室温下 搅拌4h，结束反应，将反应液倒入二氯甲烷中，抽滤，干燥，得活性酯23白色固体，直接用于下一步反应。将活性酯和NH2-PEG2-COOH(化合物24)按等当量比例溶于DMF，室温搅拌至反应结束(TLC监测)。加水，析出大量固体，抽滤，干燥，柱层析分离纯化(二氯甲烷∶甲醇＝10∶1)得白色固体化合物19，收率86％；熔点221℃；高分辨质谱分子量HRMS(ESI)理论值m/z 603.2746，found 604.2757(M+H)。
将19(1eq)，NHS(1.2eq)和EDC(1.3eq)溶于无水DMF，室温下搅拌12h，结束反应，将反应液倒入二氯甲烷中，抽滤，干燥，得活性酯化合物11白色固体，收率82％；熔点187℃；高分辨质谱分子量HRMS(ESI)理论值m/z700.2839，found 701.2842(M+1)。
偶联前体化合物13合成举例：
Figure PCTCN2017081007-appb-000035
将化合物20-1(参考中国专利CN201210382202.8)(1eq)和巯基乙酸(1eq)溶于无水DMF，加入1.2eq HBTU，3eq三乙胺以及0.1eq DMAP，室温搅拌，反应结束后，加水，抽滤，干燥得20-2淡黄色固体。再与4-(N-马来酰亚胺甲基)环己烷-1-羧酸(1eq)在DMF中室温搅拌，TLC监测原料消失，反应混 合物倾倒入冷水，产物固体过滤，干燥得化合物20，MS(ESI)656.27(M+1).将化合物20(1eq)加入NHS(1.2eq)和EDC(1.3eq)的DMF溶液室温搅拌过夜，反应完成后，经制备液相分离得活性酯化合物13，熔点201℃，收率35％(从起始原料20-1算起)；高分辨质谱分子量HRMS(ESI)理论值m/z 752.2630，found 753.2632(M+1)。
偶联前体化合物15、17合成制备方法、反应条件和投料比例与制备化合物13相同，只是将
Figure PCTCN2017081007-appb-000036
用
Figure PCTCN2017081007-appb-000037
替换；
偶联前体化合物15合成路线：
Figure PCTCN2017081007-appb-000038
得化合物21，白色固体，质谱鉴定分子量：HRMS(ESI)630.2356(M+1)。得偶联前体化合物15，白色固体，质谱鉴定分子量：HRMS(ESI)727.2431(M+1)。
偶联前体化合物17的合成路线：
Figure PCTCN2017081007-appb-000039
Figure PCTCN2017081007-appb-000040
得化合物22，白色固体，质谱鉴定分子量：HRMS(ESI)588.1819(M+1)。得偶联前体化合物17，白色固体，质谱鉴定分子量：HRMS(ESI)685.2022(M+1)。
以PD-1抗体形成的本发明中新型双功能抗体制备合成实施举例：
物质鉴定质谱仪型号：LDI-1700激光解吸电离飞行时间质谱(MALDI-TOF-MS)，生产商：美国Linear Scientific公司。
质谱确定小分子免疫激动剂的比例(偶联度)的方法如下：质谱鉴定所得偶联新型抗体产物的分子量减去原PD-1抗体分子量得增加值，计算：分子量增加值/(偶联前体小分子分子量-18)＝偶联度；(小分子化合物每偶联1个减去1个水分子的分子量)。
PD-1抗体首选为Nivolumab(BMS-936558)、Pembrolizumab(MK-3475)，AMP-514，AMP-224，Pidilizumab；但是也适用于其它来源的PD-1抗体。
Nivolumab的制备按照专利WO2006121168中制备5C4、17D8、2D3、4H1、4A11、7D3和5F4的方法；及其CN201380062005.0中公布的方法。
Pembrolizumab的制备按照专利WO2008156712(A1)中制备h409A11、及其US8354509和US8900587专利中公布的方法。
AMP-514和AMP-224的制备按照专利WO201214549专利中公布的方法。
Pidilizumab的制备按照专利WO2009014708和WO2009114335专利中公布的方法。
PD-1抗体的制备方法也可采用本领域公知的其它公开方法，如在中国专利CN201410838610.9；CN201310199947.5；CN201380079581.6；CN201480011008.6；CN201310258289.2的中公开的方法。
PD-1抗体也可以市场购买为来源，比如购自美国BioXCell公司PD-1抗体，名称：anti h PD-1(h CD279)IgG1，含量95％；北京科昕生物科技有限公司抗PD-1 全人单克隆抗体。本发明新型抗体合成方法中的PD-1抗体来源不局限于此。
以下PD-1抗体的反应浓度均为1当量浓度(1eq，相对于其它反应物)。
新型抗体10的制备：
偶联前体小分子化合物1的合成制备方法请参考中国专利(CN201210382202.8)。
将化合物1(10eq当量浓度)和PD-1抗体(Nivolumab)混合于1mL的DMSO溶剂中，混合物加入三乙胺0.1mL，与5℃至20℃搅拌反应12小时。反应混合物与10mL的0℃纯水混合，摇匀，用10kD的生物滤膜过滤除去小分子，用纯水洗脱产物，洗脱液冷冻干燥得新型抗体10(收率60％)。质谱鉴定分子量增加为1167；计算偶联度为3。(1167/386＝3.02)。
新型抗体12的制备：
将化合物11(30eq当量浓度)和PD-1抗体(Nivolumab)混合于适量DMSO溶剂中，混合物加入三乙胺调节pH值到8，与5℃至20℃搅拌反应12小时。反应混合物与10倍体积的0℃纯水混合，摇匀，用10kD的生物滤膜过滤除去小分子，用纯水冲洗脱产物，洗脱液冷冻干燥得新型抗体12(收率72％)。质谱鉴定分子量增加为2931；计算偶联度为5。(2931/585.6＝5)。
(在新型抗体12的制备方法中，将Nivolumab替换为BioXCell公司PD-1抗体：anti h PD-1(h CD279)IgG1，得到新型抗体12-2，其制备过程和所得新型抗体12-2的结果与新型抗体12相同)。
新型抗体14的制备：
将化合物13(40eq当量浓度)和PD-1抗体(AMP-514)混合于适量DMSO溶剂中，混合物加入三乙胺调节pH值到8，与5℃至20℃搅拌反应12小时。反应混合物与10倍体积的0℃纯水混合，摇匀，用10kD的生物滤膜过滤除去小分子，用纯水冲洗脱产物，洗脱液冷冻干燥得新型抗体14(收率66％)。质谱鉴定分子量增加为1917；计算偶联度为3。
新型抗体16的制备：
将化合物15(40eq当量浓度)和PD-1抗体(Pembrolizumab)混合于适量DMSO溶剂中，混合物加入三乙胺调节pH值到8，与5℃至20℃搅拌反应12小时。 反应混合物与10倍体积的0℃纯水混合，摇匀，用10kD的生物滤膜过滤除去小分子，用纯水冲洗脱产物，洗脱液冷冻干燥得新型抗体16(收率73％)。质谱鉴定分子量增加为2377；计算偶联度约为4。
新型抗体18的制备：
将化合物17(40eq当量浓度)和PD-1抗体(AMP-224)混合于适量DMSO溶剂中，混合物加入三乙胺调节pH值到8，与5℃至20℃搅拌反应12小时。反应混合物与10倍体积的0℃纯水混合，摇匀，用10kD的生物滤膜过滤除去小分子，用纯水冲洗脱产物，洗脱液冷冻干燥得新型抗体18(收率57％)。质谱鉴定分子量增加为1143；计算偶联度约为2。
本发明新型抗体的免疫激活实验方法(小鼠)：
1.取Balb/C小鼠脾淋巴细胞，按1x106/ml/孔铺板
2.优选抗体浓度：按0.1μM加药刺激24h。
3.收集上清并用ELISA方法检测IL-6/IL-12(效果如图1、2所示)。
本发明中小分子免疫激动剂的免疫激活实验方法(小鼠)：
1.取Balb/C小鼠脾淋巴细胞，按1x106/ml/孔铺板
2.选取小分子免疫激动剂的浓度：按0.1、10、20μM加药刺激24h。
3.收集上清并用ELISA方法检测IFN-γ/IL-6(效果如图6、7所示)。
本发明新型抗体的免疫激活实验方法(人)：
1.取自愿者外周单核细胞，分离出T细胞(悬浮细胞)于37℃，5％CO2含血清培养基培养7天，按1x106/ml/孔铺板
2.优选抗体浓度：按0.1μM加药刺激24h。
3.收集上清并用ELISA方法检测IFN-γ(如图3所示)。
本发明新型抗体的抗肿瘤效果实验方法：
将6-8周龄的Balb/C小鼠随机分为7组。在小鼠背部皮下种植2.5x105个4T1肿瘤细胞。分别用PBS空白对照、10mg/kg的PD-1抗体对照给药；对照药和新型抗体10、12、14、16、18的给药剂量为10mg/kg、体积100μL；各组给药方式为腹腔注射。在种植肿瘤后的第1天，第7、15、22、29天各组分别给药。当肿瘤达到1500mm3或者大于体重的15％时将小鼠安乐死。肿瘤抑制结果如图4所 示。可见本发明中新型抗体处理组具有显著提高的抗肿瘤效应。
本发明新型抗体的促进T细胞增值实验方法：
取自愿者外周单核细胞，分离出T细胞(悬浮细胞)于37℃，5％CO2含血清培养基培养7天后，换无血清培养基同时加入本发明的新型抗体和PD-1(对照)(浓度均为1μM)，3天后CCK-8细胞染色方法检测。可见本发明新型抗体可以通过加强阻断PD-1和激活天然免疫促进T细胞增值。结果如图5所示。
本发明新型抗体的PD-1抗体功能特征实验方法(用PD-1的特异性二抗检测PD-1抗体)：
1.抽取健康志愿者外周血
2.取得外周血于离心管中，800g，30min。分离血清和血细胞。
3.获得血细胞后重悬于生理盐水中，轻轻沿壁加入至外周血淋巴细胞分离液上层。
4.800g离心30min.吸取白膜层(外周淋巴细胞层)。
5.将分离得到的外周血淋巴细胞重悬于生理盐水中900g，10min。洗涤2次。
6.计数分离获得的外周血淋巴细胞。
7.取1x106细胞，100ul生理盐水重悬，分别用PD-1抗体，本发明新型抗体10、12、16，和IgG1(对照)，37度孵育30min。
8.生理盐水洗涤3次，用100ul生理盐水重悬，加入Anti-PD-1-FITC的二抗，室温孵育30min。
9.生理盐水洗涤，流式上机检测。
结果见如图8、9、10、11所示：与对照组以及未偶联的组比较可以发现，本发明的新型抗体的PD-1抗体功能特征保持完好。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应该以权利要求的保护范围为准。

Claims (10)

  1. 一种小分子免疫激动剂偶联PD‐1抗体的新型抗体，其特征在于，所述新型抗体由小分子免疫激动剂和PD‐1抗体偶联反应得到，所述新型抗体包括以下通式[Ⅰ]的化合物：
    Figure PCTCN2017081007-appb-100001
    式[Ⅰ]中：X1表示OH或SH；R1表示烷氧基或烷基胺基，X2表示链接基团；其中抗体部分与小分子免疫激动剂组成部分是由小分子免疫激动剂偶联前体和所述抗体共价化合链接形成，m为小分子激动剂的数目(m定义为偶联度)，为1～10的数字；抗体部分指为抗PD-1的单抗(IgG1、IgG2或IgG4)；所述小分子免疫激动剂偶联前体定义为与抗体进行共价化合时的小分子免疫激动剂的活化形式。
    当所述小分子免疫激动剂偶联前体为式1的化合物时：
    Figure PCTCN2017081007-appb-100002
    所述X2表示硫代羰基基团：
    Figure PCTCN2017081007-appb-100003
    当所述小分子免疫激动剂偶联前体为式2-1、2-2、2-3的化合物时：
    Figure PCTCN2017081007-appb-100004
    所述X2表示如下基团：
    Figure PCTCN2017081007-appb-100005
    当所述小分子免疫激动剂偶联前体为式3的化合物时：
    Figure PCTCN2017081007-appb-100006
    式3中，u为0～12的整数；
    X2表示如下基团：
    Figure PCTCN2017081007-appb-100007
    其中u为0～12的整数；
    当所述小分子免疫激动剂偶联前体为式4的化合物时：
    Figure PCTCN2017081007-appb-100008
    X2表示如下基团：
    Figure PCTCN2017081007-appb-100009
    其中PEG为聚乙二醇基团
    Figure PCTCN2017081007-appb-100010
    如二聚乙二醇基团为
    Figure PCTCN2017081007-appb-100011
    三聚乙二醇基团为
    Figure PCTCN2017081007-appb-100012
    四聚乙二醇基团为
    Figure PCTCN2017081007-appb-100013
  2. 根据权利要求1所述的小分子免疫激动剂偶联PD-1抗体的新型抗体，其特征在于：所述PD-1抗体包括各种抗人PD-1蛋白的单抗(IgG1、IgG2或IgG4)，所述PD-1蛋白的序列优先选择如下：
    1MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTS61ESFVLNWYR70MSPSNQTDKLAAFPEDR86SQPGQDCRFRVTQLPNGRDFHMSVVRARRND118SGTYLCGAISLAPKAQIKE136SLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGS18 1LVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVP241CVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL；
    所述PD-1抗体针对该序列为抗原的表位序列。
  3. 根据权利要求1所述的小分子免疫激动剂偶联PD-1抗体的新型抗体，其特征在于：所述的PD-1抗体为Nivolumab、Pembrolizumab、AMP-514、AMP-224、Pidilizumab或其它anti h PD‐1(h CD279)IgG1。
  4. 根据权利要求1所述的小分子免疫激动剂偶联PD-1抗体的新型抗体，其特征在于：当小分子免疫激动剂为式1、2-1、2-2、2-3、4代表的化合物时，所述新型抗体的典型代表结构式如下:
    Figure PCTCN2017081007-appb-100014
    Figure PCTCN2017081007-appb-100015
    其中m为1到10之间的数。
  5. 根据权利要求4所述的小分子免疫激动剂偶联PD-1抗体的新型抗体，其特征在于：当小分子免疫激动剂为式1、2-1、2-2、2-3、4代表的化合物时，所述新型抗体的典型代表10、12、14、16、18的结构式如下:
    Figure PCTCN2017081007-appb-100016
    Figure PCTCN2017081007-appb-100017
    Figure PCTCN2017081007-appb-100018
  6. 一种小分子免疫激动剂偶联前体化合物，其特征在于：用于制备如权利要求5中的新型抗体的典型代表12、14、16、18，对应包括以下结构式11、13、15、17的化合物：
    Figure PCTCN2017081007-appb-100019
    Figure PCTCN2017081007-appb-100020
  7. 用于合成如权利要求6所述的小分子免疫激动剂偶联前体化合物的小分子免疫激动剂化合物，其特征在于：所述小分子免疫激动剂化合物为包括如下所示结构式19、20-2、20、21、22的化合物或它们的盐：
    Figure PCTCN2017081007-appb-100021
    Figure PCTCN2017081007-appb-100022
  8. 根据权利要求1---7中任一项所述的新型抗体和小分子化合物或它们的盐制成的适用于各种治疗药物、或与其他药物制成复方药物、协同药物，或制成药学上可接受的载体的复合物或结合物。
  9. 使用如权利要求8中所述的药物，在免疫抑制的解除和激发、抗体制备、抗病毒、糖尿病、和肿瘤免疫治疗中的应用。
  10. 如权利要求9中所述的药物的应用，用药方法为腹腔注射、皮下注射、肌肉注射和静脉注射或口服；或者可用于和免疫细胞(如树突状细胞、自然杀伤细胞NK、淋巴细胞、单核/巨噬细胞、粒细胞等)共培养后，分离免疫细胞体内回输的方法。
PCT/CN2017/081007 2016-08-15 2017-04-19 小分子免疫激动剂偶联pd-1抗体的新型抗体及其在抗肿瘤中的应用 Ceased WO2018032793A1 (zh)

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