Beta-glucan antibody drug conjugate, preparation method and immune anti-tumor application

Abstract

The invention provides a beta-glucan antibody drug conjugate as well as a preparation method and immune anti-tumor application thereof. Specifically, the invention provides a conjugate or a pharmaceutically acceptable salt thereof, the conjugate is shown as a formula (I), Ab is an antibody part, and the antibody is an antibody targeting an immune checkpoint or an antibody targeting a signal transduction receptor; bG is a beta-glucan moiety; l is a connecting arm for connecting the antibody and beta-glucan; and subscript n is 1 to 10. The conjugate disclosed by the invention not only can play an immune regulation role of beta-glucan and a targeting antibody, but also can play a bridging role on immune cells and tumor cells, and anti-tumor experiments find that the conjugate has remarkable anti-tumor activity and can be used for developing anti-tumor drugs. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-tumor therapeutic drugs, and specifically relates to a β-glucan antibody-drug conjugate, a preparation method and immune anti-tumor application. Background Art

[0002] Immune checkpoint inhibitors such as PD-1 / PD-L1 have been successful in the treatment of a variety of tumors, but these immune suppression effects only occur in a subset of patients (about 20%) with specific tumor histological characteristics. Moreover, many patients who are initially sensitive to PD-1 / PD-L1 antibodies will eventually develop resistance, leading to tumor recurrence and immune-related adverse reactions unique to tumor immunotherapy. Therefore, it is very necessary to develop therapeutic antibody drugs that inhibit the PD-L1 / PD-1 pathway to improve patient resistance, improve clinical treatment effects, reduce immune-related adverse reactions, and prolong overall survival of patients.

[0003] In summary, there is an urgent need to develop a new anti-tumor drug in this field. Summary of the invention

[0004] The purpose of the present invention is to provide a β-glucan antibody drug conjugate (Antibody-beta-glucan-conjucates, AGC), in particular, a conjugate of an antibody and a glucan immunomodulator for immunotherapy, such as an anti-PD-L1 antibody and β-glucan conjugate, and a preparation method and application thereof (such as application in anti-tumor).

[0005] In the first aspect of the present invention, a conjugate or a pharmaceutically acceptable salt thereof is provided, wherein the conjugate is represented by formula (I):

[0006]

[0007] in,

[0008] Ab is an antibody portion, and the antibody is an antibody targeting an immune checkpoint or an antibody portion targeting a receptor for signal transduction;

[0009] BG is the β-glucan part;

[0010] L is a linker that connects the antibody and β-glucan; and

[0011] The subscript n is 1 to 10.

[0012] In another preferred embodiment, the antibody includes: a chimeric antibody, a humanized antibody, a fully human antibody, a nanobody, or a combination thereof.

[0013] In another preferred embodiment, the antibody comprises one or more RSCRA The amino acid residues of the group, where R SCRA Refers to the reactive group in the amino acid side chain (such as -NH2 or -SH in the amino acid side chain).

[0014] In another preferred embodiment, R SCRA It is -NH2 or -SH.

[0015] In another preferred embodiment, R SCRA The amino acid residue of the group is selected from the group consisting of a lysine residue and a cysteine ​​residue.

[0016] In another preferred embodiment, the amino acid sequence of the antibody contains one or more lysine residues or cysteine ​​residues.

[0017] In another preferred embodiment, the R SCRA The amino acid residue of the group (preferably, a lysine residue or a cysteine ​​residue) is the site of attachment to the rest of the conjugate.

[0018] In another preferred embodiment, some or all of the R-containing SCRA The amino acid residue of the group forms a bond with the rest of the conjugate.

[0019] In another preferred embodiment, the antibody portion refers to the R that does not include the R that has been connected to the rest of the conjugate. SCRA of the antibody portion.

[0020] In another preferred embodiment, the immune checkpoint is selected from the following group: PD-1 / PD-L1, CTLA-4, LAG-3, TIM-3 and TIGIT.

[0021] In another preferred embodiment, the signal transduction receptors include: EGFR.

[0022] In another preferred embodiment, the antibody is an antibody targeting an immune checkpoint.

[0023] In another preferred embodiment, the immune checkpoint is PD-1 / PD-L1.

[0024] In another preferred embodiment, the antibody targeting the immune checkpoint is selected from the following group: PD-L1 antagonist, programmed death 1 protein (PD-1) antagonist, cytotoxic T lymphocyte antigen (CTLA-4) antagonist, lymphocyte activation gene-3 (LAG-3) antagonist, T cell immunoglobulin-3 (TIM-3) antagonist, T cell immunoglobulin and ITIM domain protein (TIGIT) antagonist, or a combination thereof.

[0025] In another preferred embodiment, the antibody targeting the immune checkpoint is selected from the following group: anti-PD-L1 antibody, anti-PD-1 antibody, or a combination thereof.

[0026] In another preferred embodiment, the antibody targeting the immune checkpoint is an anti-PD-L1 antibody.

[0027] In another preferred embodiment, the antibody targeting the receptor for signal transduction comprises an epidermal growth factor receptor antagonist (EGFR antagonist).

[0028] In another preferred embodiment, the antibody is selected from the group consisting of ipilimumab, pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, cergutuzumab, ibritumomab tiuxetan, rituximab, tositumomab, gemtuzumab, alemtuzumab, cetuximab, panitumumab, depatuxizumab, sibrotuzumab, codrituzumab, trastuzumab, patritumab, figitumumab, ganitumab, cantuzumab, bavituximab, palivizumab, bevacizumab, or a combination thereof.

[0029] In another preferred embodiment, the antibody (anti-PD-1 antibody / anti-PD-L1 antibody) is selected from the following group: pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, or a combination thereof.

[0030] In another preferred embodiment, the antibody (anti-PD-L1 antibody) is selected from the following group: atezolizumab, avelumab, durvalumab, or a combination thereof.

[0031] In another preferred embodiment, the antibody is an anti-mouse PD-L1 antibody (αPD-L1, 10F.9G2).

[0032] In another preferred example, the molecular weight of the anti-mouse PD-L1 antibody (αPD-L1, 10F.9G2) is 146671.1 Da.

[0033] In another preferred embodiment, the β-glucan is connected to other parts of the conjugate via its reducing end.

[0034] In another preferred embodiment, the β-glucan moiety is a moiety formed by β-glucan losing its hemiacetal hydroxyl group or hydroxyl hydrogen (for example, due to reaction with a reactive group such as NH2 or COOH).

[0035] In another preferred embodiment, the β-glucan is β-1,3-glucan containing β-1,6 branches (ie, β-1,3 / 1,6-glucan).

[0036] In another preferred embodiment, the β-glucan is water-soluble β-glucan.

[0037] In another preferred embodiment, the molecular weight of the β-glucan is 1-800 kDa; preferably, 1-30 kDa; more preferably, 1-10 kDa.

[0038] In another preferred embodiment, the molecular weight of the β-glucan is 2-6 kDa.

[0039] In another preferred embodiment, the β-glucan is derived from brown algae or fungi; preferably, it is derived from brown algae.

[0040] In another preferred embodiment, the β-glucan is derived from Antarctic sea antlers.

[0041] In another preferred embodiment, the Antarctic sea antler is selected from one or more of Durvillaea antarctica, D.poha, D.chathamensis, D.potatorum, D.willana and D.amatheiae.

[0042] In another preferred embodiment, the Antarctic sea antlers are mainly derived from Durvillaea antarctica.

[0043] In another preferred embodiment, n represents the average number of β-glucans coupled to each antibody.

[0044] In another preferred embodiment, n can be an integer or a non-integer.

[0045] In another preferred embodiment, n is 1 to 5; preferably, n is 1 to 4; more preferably, n is 1.5 to 2.5; most preferably, n is 2 to 3.

[0046] In another preferred embodiment, L is a linker having 5 to 90 chain atoms (preferably 10 to 80, preferably 20 to 70, more preferably 30 to 60, and optimally 40-50) on the main chain, 0 to 3 rings, and 0 to 5 (preferably 2, 3, 4 or 5, optimally 2 or 3) linking groups formed by reaction.

[0047] In another preferred embodiment, L is a linker composed of 5 to 90 chain atoms (preferably 10 to 80, preferably 20 to 70, more preferably 30 to 60, and optimally 40-50) divalent groups, 0 to 3 3 to 16-membered rings, and 0 to 5 (preferably 2, 3, 4 or 5, optimally 2 or 3) linking groups formed by reaction; wherein the divalent group is selected from the group consisting of -C(R)2-, -N(R)-, -S-, -O-, -CO-, -SO- and -SO2- (preferably, the divalent group is selected from the group consisting of -CH2-, -NH-, -S-, -O-, -CO-, -SO- and -SO2-); wherein R is independently H or C 1-4 Alkyl, with the additional proviso that any two adjacent divalent radicals are not simultaneously O.

[0048] In another preferred embodiment, the linking group formed by the reaction includes: a dextran linking group, an antibody linking group and 0, 1, 2 or 3 linker coupling linking groups.

[0049] In another preferred embodiment, the glucan linking group is a linking group (such as -NH-, -CO-) formed by the reaction of a reactive group (such as NH2 or COOH) with a hydroxyl group in the glucose group in β-glucan.

[0050] In another preferred embodiment, the antibody linking group is an antibody containing R SCRA The amino acid residue R SCRA Groups (such as -NH2 and -SH) that can interact with the R SCRA The groups react to form active groups (such as COOH or ) The linking group formed after the reaction (preferably, R SCRA When it is -NH2, the linking group is -CONH-, and when RSCRA is -SH, the linking group is

[0051] In another preferred embodiment, the linker coupling linking groups are independently selected from the following groups: -N(R)-, -CO-, -N(R)CO-, -COO-, linking groups formed by bioorthogonal reactions, -SH and The linking group formed by the reaction.

[0052] In another preferred embodiment, the linking group formed by the bioorthogonal reaction includes: DBCO (dibenzocyclooctene) or cyclopropanecyclooctyne (BCN) reacts with -N3 to form a group, C 3-8 A group formed by a cycloolefin (such as trans-cyclooctene (TCO) or cyclopropene) and 1,2,4,5-tetrazine.

[0053] In another preferred embodiment, the reactive group is selected from the following group: amino group (-NH2), amide group (-CONH2), maleimide group, and bioorthogonal reactive group.

[0054] In another preferred embodiment, the bioorthogonal reactive group includes: trans-cyclooctenyl, tetrazine, dibenzocyclooctyne or azide.

[0055] In another preferred embodiment, the linking group formed by the bioorthogonal reaction comprises:

[0056] In another preferred embodiment, the chain atoms are selected from the group consisting of C, N, O and S.

[0057] In another preferred embodiment, the ring may be a carbocyclic ring or a heterocyclic ring containing one or more heterocyclic atoms selected from N, O and S.

[0058] In another preferred embodiment, the rings are each independently an aromatic ring or a non-aromatic ring.

[0059] In another preferred embodiment, the rings are each independently a monocyclic ring, a condensed ring or a spirocyclic ring.

[0060] In another preferred embodiment, the ring has 3 to 16 (preferably 3 to 10, more preferably 3 to 6) ring atoms.

[0061] In another preferred embodiment, any two rings among the rings are adjacent (ie, the two rings are connected by a single bond) or separated (ie, there is a chain atom between the two rings).

[0062] In another preferred embodiment, L is as shown in Formula II-a;

[0063] -W BG -L 1 -W 1 -L 2 -W 2 -L 3 -W Ab -(II-a);

[0064] -W BG -L 1 -W 1 -L 4 -W Ab -(II-b);

[0065] in,

[0066] W BG is the end connected to BG, and W BG is a dextran linking group (preferably, W BG Selected from the group consisting of: none, -NH-, -CO-);

[0067] W Ab is an antibody linking group;

[0068] L 1 None or -(M 13 ) o1 -(M 11 -M 12 ) s1 -(M 14 ) o2 -; among them, each M 13 and M 14 are independently selected from the group consisting of none, -CO-, -N(R)-, -O-, -S- and -C(R)2-, subscripts o1 and o2 are each independently 0, 1, 2, 3 or 4, each M 11 are independently selected from the group consisting of -C(R)2- and -C(R)2C(R)2-, each M 12 Independently selected from the group consisting of none, -C(R)2-, -C(R)2C(R)2-, O and S, subscript s1 is an integer from 1 to 20 (preferably, an integer from 1 to 15, more preferably, an integer from 1 to 10, and most preferably, 1, 2, 3 or 4);

[0069] W 1 is no or a connector coupling linking group (preferably, W 1 Selected from the group consisting of none, NH, CO, -NHCO-, and -COO-);

[0070] L 2 None or -(M 24 ) p1 -(M 21 -M 22 -M 23 ) s2 -(M 25 ) p2 -; among them, M 21 and M 22 Each independently is none or C(R)2, M 23 Each is independently selected from the following group: O, S and C (R) 2, subscript s2 is an integer from 1 to 10 (preferably, an integer from 2 to 8, more preferably, 3, 4, 5, 6 or 7), each M 24 and M 25independently selected from the group consisting of none, -CO-, -N(R)-, -O-, -S-, -C(R)2-, optionally substituted phenyl, optionally substituted 5- or 6-membered heteroaryl, optionally substituted C 3-6 Carbocyclyl and optionally substituted 4- to 6-membered heterocyclyl, subscripts p1 and p2 are each independently 0, 1, 2 or 3;

[0071] W 2 is without or with a linker coupling linking group (preferably, a linking group formed by a bioorthogonal reaction);

[0072] L 3 None or *-(M 34 ) q1 -(M 31 -M 32 -M 33 ) s3 -(M 35 ) q2 -; where * represents the same as W 2 The connection position, M 31 and M 32 Each independently is none or C(R)2, M 33 Each is independently selected from the following group: O, S and C (R) 2, subscript s3 is an integer from 1 to 15 (preferably, an integer from 2 to 12, more preferably, 2, 3, 4, 5 or 6), each M 34 and M 35 Independently selected from the group consisting of none, -CO-, -N(R)-, -O-, -S-, -C(R)2-, subscripts q1 and q2 are each independently 0, 1, 2, 3, 4 or 5;

[0073] L 4 None or -(M 43 ) r1 -(M 41 -M 42 ) s4 -(M 44 ) r2 -; among them, M 41 Each is independently selected from the following group: none, -C(R)2-, -C(R)2C(R)2-, M 42 Each is independently selected from the group consisting of none, O and S, subscript s4 is an integer from 0 to 10 (preferably, an integer from 1 to 5, more preferably, 1 or 2), each M 43 and M 44 are independently selected from the group consisting of none, -CO-, -N(R)-, -O-, -S-, -C(R)2-, optionally substituted C 3-6carbocyclyl, optionally substituted 4- to 6-membered heterocyclyl, optionally substituted phenyl, and optionally substituted 5- or 6-membered heteroaryl, subscripts R1 and R2 are each independently 0, 1, 2 or 3;

[0074] R is independently selected from the following group: H, C 1-4 Alkyl (preferably, R is H).

[0075] In another preferred embodiment, L 1 , L 2 and L 3 At least one of them is not nothing.

[0076] In another preferred embodiment, L 1 and L 4 At least one of them is not nothing.

[0077] In another preferred embodiment, W BG It is -NH-.

[0078] In another preferred embodiment, W Ab It is -CONH-.

[0079] In another preferred embodiment, W Ab for

[0080] In another preferred embodiment, -(M 13 ) o1 - is -N(R)CO-, preferably, -NHCO-. In another preferred embodiment, -(M 14 ) o2 - is -CON(R)-, preferably, -CONH-. In another preferred embodiment, 1-(M 11 -M 12 ) s1 -for-(C(R)2) s1 -, preferably -(CH2) s1 -.

[0081] In another preferred embodiment, L 1 -NHCO-(CH2) s1 -CONH-, wherein subscript s1 = 1, 2, 3, 4, 5, 6, 7 or 8 (preferably, subscript s1 = 2, 3, 4, 5 or 6).

[0082] In another preferred embodiment, W 1 -NHCO-, where NH is L 1 One end of the connection.

[0083] In another preferred embodiment, -(M 24 ) p1 - is none. In another preferred embodiment, -(M21 -M 22 -M 23 ) s2 - is -(C(R)2C(R)2O) s2 -; preferably, -(CH2CH2O) s2 -. In another preferred embodiment, -(M 25 ) p2 -for-M 25 -; and M 25 is selected from the group consisting of none, -C(R)2-, optionally substituted phenyl, optionally substituted 5- or 6-membered heteroaryl, optionally substituted C 3-6 carbocyclic group and optionally substituted 4 to 6 membered heterocyclic group (preferably, M 25 is phenyl).

[0084] In another preferred embodiment, L 2 -(CH2CH2O) s2 -M 25 -, wherein the subscript s2 is an integer from 1 to 10 (preferably, an integer from 2 to 8, more preferably, 3, 4, 5, 6 or 7), M 25 is selected from the group consisting of none, -C(R)2-, optionally substituted phenyl, optionally substituted 5- or 6-membered heteroaryl, optionally substituted C 3-6 carbocyclic group and optionally substituted 4 to 6 membered heterocyclic group (preferably, M 25 is phenyl).

[0085] In another preferred embodiment, W 2 for

[0086] In another preferred embodiment, -(M 34 ) q1 - is -OCON(R)-; preferably, -OCONH-. In another preferred embodiment, -(M 31 -M 32 -M 33 ) s3 - is -(C(R)2C(R)2O) s3 -; preferably, -(CH2CH2O) s3 -. In another preferred embodiment, -(M 35 ) q2 -for-(C(R)2) 1-2 Preferably, -(CH2) 1-2 -.

[0087] In another preferred embodiment, L 3 -OCONH-(CH2-CH2-O) s3 -(CH2) 1-2-, preferably wherein -OCONH- is 2 At the connected end, the subscript s3 is 2, 3, 4, 5 or 6.

[0088] In another preferred embodiment, -(M 43 ) r1 - for C 3-6 In another preferred embodiment, -(M 41 -M 42 ) s4 -for-(C(R)2) s4 -; preferably, -(CH2) s4 -. In another preferred embodiment, -(M 44 ) r2 - is none.

[0089] In another preferred embodiment, L 4 None or -cyclohexyl-(CH2) s4 -.

[0090] In another preferred embodiment, the conjugate is as shown in formula III-a or III-b

[0091]

[0092]

[0093] wherein BG, Ab, subscripts s1, s2, s3 and s4 are as defined above.

[0094] In another preferred embodiment, in Formula III-a or Formula III-b, Ab represents an anti-PD-L1 antibody or an anti-PD-1 antibody, preferably, an anti-PD-L1 antibody.

[0095] In another preferred embodiment, in formula III-a or formula III-b, BG is a β-glucan portion, and the β-glucan is derived from Antarctic sea antlers (preferably, the Antarctic sea antlers are selected from one or more of Durvillaea antarctica, D.poha, D.chathamensis, D.potatorum, D.willana and D.amatheiae).

[0096] In another preferred embodiment, s1 is an integer from 1 to 20, and / or s2 is an integer from 1 to 10, and / or s3 is an integer from 1 to 15, and / or s4 is an integer from 0 to 10.

[0097] In another preferred embodiment, s1=1, 2, 3, 4, 5, 6, 7 or 8, and / or s2=3, 4, 5, 6 or 7, and / or s3=2, 3, 4, 5 or 6, 8, and / or s4=1 or 2.

[0098] In the second aspect of the present invention, a pharmaceutical composition is provided, comprising (a) the conjugate or a pharmaceutically acceptable salt thereof as described in the first aspect, and (b) a pharmaceutically acceptable carrier or excipient.

[0099] In the third aspect of the present invention, there is provided a use of the conjugate or a pharmaceutically acceptable salt thereof as described in the first aspect, or a pharmaceutical composition comprising the conjugate or a pharmaceutically acceptable salt thereof in the preparation of an anti-tumor or cancer treating drug.

[0100] In another preferred embodiment, the cancer is selected from the group consisting of melanoma, colorectal cancer, colon cancer, lung cancer, kidney cancer, liver cancer, breast cancer, or a combination thereof.

[0101] In another preferred embodiment, the cancer is colon cancer.

[0102] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0103] Figure 1 Shown is the SDS-PAGE analysis of β-1,3 / 1,6-glucan PD-L1 antibody conjugate.

[0104] Figure 2 The drug-antibody conjugate ratio of β-1,3 / 1,6-glucan PD-L1 antibody conjugate is shown.

[0105] Figure 3 Shows the binding effect between β-1,3 / 1,6-glucan PD-L1 antibody conjugate and PD-L1 antigen antibody (ELISA).

[0106] Figure 4 The binding ability of β-1,3 / 1,6-glucan PD-L1 antibody conjugate to cell surface PD-L1 antigen (MC38, FACS) is shown.

[0107] Figure 5 Showing the bridging effect of β-1,3 / 1,6-glucan PD-L1 antibody conjugate on MC38 melanoma cells.

[0108] Figure 6 Showing the bridging effect of β-1,3 / 1,6-glucan PD-L1 antibody conjugate on B16F10 colon cancer cells.

[0109] Figure 7The anti-tumor effect of β-1,3 / 1,6-glucan PD-L1 antibody conjugate on the MC38 syngeneic tumor model was shown. A. Treatment with αPD-L1-DAG, anti-PD-L1 antibody (αPD-L1) alone,

[0110] Inhibitory activity of β-1,3 / 1,6-glucan DAG alone and anti-PD-L1 antibody / DAG combination therapy on MC38 tumor growth in mice. B. Tumor weight at the end time point. The percentage on the graph represents the tumor inhibition rate. C. Effects of αPD-L1-DAG treatment, anti-PD-L1 antibody alone, DAG alone, and anti-PD-L1 antibody / DAG combination therapy on mouse body weight. D: Tumor size at the end time point. × on the graph represents tumor disappearance. Statistically significant differences were analyzed by unpaired two-tailed T test, One-way ANOVA analysis of variance with Tukey's multiple comparison test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001VS PBS group; ns: no statistical difference.

[0111] Figure 8 Shown is the effect of β-1,3 / 1,6-glucan PD-L1 antibody conjugate on immune cells in the MC38 syngeneic tumor model.

[0112] Fig. 9 Shown are the effects of β-1,3 / 1,6-glucan PD-L1 antibody conjugate on immune cell infiltration, chemokine, and cytokine expression in the MC38 syngeneic tumor model 24 hours after administration.

[0113] Fig.10 The antitumor effect of β-1,3 / 1,6-glucan PD-L1 antibody conjugate on the CT26 syngeneic tumor model is shown. A. Inhibitory activity of αPD-L1-DAG treatment and anti-PD-L1 antibody (αPD-L1) alone on the growth of CT26 tumors in mice. B. Tumor weight at the end time point. The percentages on the graph represent the inhibition rate of the tumor. C. Tumor size at the end time point. Statistically significant differences were analyzed by unpaired two-tailed T test, One-way ANOVA analysis of variance with Tukey's multiple comparison test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001VS PBS group; ns: no statistical difference.

[0114] Fig.11 The in vivo anti-tumor effects of the conjugates αPD-1-DAG, Cetu-DAG, BYGLS2-PD-L1, and αPD-L1-DAG-SMCC in Example 11 are shown.

[0115] Fig.12 H NMR spectra of DAG, DAG-ADH and DAG-ADH-Tz are shown. DETAILED DESCRIPTION

[0116] After long-term and in-depth research, the inventor unexpectedly discovered that the conjugate formed by β-glucan and antibodies (such as anti-PD-L1 antibodies) can not only play the immunomodulatory role of β-glucan and PD-L1 antibodies, but also play a bridging role between immune cells and tumor cells. Anti-tumor experiments have found that it has significant anti-tumor activity, especially significantly better anti-tumor effect than the combination of β-glucan and antibodies, and can be used for the development of anti-tumor drugs. Based on this, the inventor completed the present invention.

[0117] Specifically, the present invention couples β-glucan (such as β-1,3 / 1,6-glucan DAG) with an antibody (such as an anti-PD-L1 antibody (αPD-L1)) through a connecting arm to obtain a β-glucan antibody conjugate (AGC, such as αPD-L1-DAG). The conjugate (AGC) of the present invention has been experimentally verified to maintain the antigen recognition ability of the original anti-PD-L1 antibody and has good selectivity. The AGC obtained has been verified by anti-tumor experiments to have strong anti-tumor activity, activate innate immunity and adaptive immunity, and improve the tumor microenvironment. It is expected to be used in the development of therapeutic drugs for various cancers (such as lymphoma, melanoma, colorectal cancer, colon cancer, lung cancer, kidney cancer, gastric cancer, liver cancer, breast cancer, etc.), and has good application prospects and value.

[0118] the term

[0119] Unless otherwise defined, the following terms used in the specification and claims have the meanings commonly understood by those skilled in the art.

[0120] As used herein, "β-glucan antibody drug conjugate", Antibody-beta-glucan-conjucates (AGC), refers to the conjugate as described in the first aspect of the present invention. "β-glucan antibody drug conjugate" herein may also be referred to as "the conjugate of the present invention", "β-glucan antibody conjugate", "β-1,3 / 1,6-glucan antibody conjugate" or "β-glucan and antibody drug conjugate". In some embodiments, the β-glucan antibody drug conjugate of the present invention is a β-1,3 / 1,6-glucan PD-L1 antibody conjugate.

[0121] As used herein, when referring to glucan, the term "reducing end" refers to the end having a free hemiacetal hydroxyl group or hemiketal group.

[0122] As used herein, the term "PD-L1" includes isoforms, mammalian forms, such as human PD-L1, murine PD-L1, species homologs of human PD-L1, and analogs that contain at least one common epitope with PD-L1.

[0123] In the present invention, the term "anti-PD-L1 antibody" refers to a mouse antibody that can specifically bind to PD-L1.

[0124] The term "alkyl", by itself or as part of another substituent, refers, unless otherwise indicated, to a straight or branched chain hydrocarbon radical (i.e., C 1-4 represents 1 to 4 carbons). Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, and the like.

[0125] As used herein, the term "alkylene", by itself or as part of another substituent, refers to a divalent group derived from an alkane, such as -CH2-. Alkylene groups typically have 1 to 4 carbon atoms (i.e., C 1-4 Examples of the alkylene group include, but are not limited to, methylene and ethylene.

[0126] Unless otherwise indicated, the term "aryl" represents a polyunsaturated (usually aromatic) hydrocarbon radical, which can be a monocyclic or polycyclic (maximum three rings) fused together or covalently attached. Typically, aryl has 6-10 ring atoms. The term "heteroaryl" refers to an aryl (or ring) containing 1 to 5 heteroatoms (preferably only containing N heteroatoms) selected from N, O and S, wherein nitrogen and sulphur atoms are optionally oxidized, and nitrogen atom is optionally quaternized. Typically, heteroaryl has 5-10 ring atoms, i.e., 5-10 yuan heteroaryl, preferably, has 5-6 ring atoms, i.e., 5-6 yuan heteroaryl, and contains 1, 2, 3 or 4 heteroatoms. Heteroaryl can be connected to the rest of the molecule by heteroatoms. The non-limiting examples of aryl include phenyl, naphthyl and biphenyl, and the non-limiting examples of heteroaryl include pyridyl etc.

[0127] In this article, the bonds represented by dashed lines or keys marked with a wavy line Represents the site at which a group (or fragment or part) is attached to the rest of the moiety.

[0128] In this document, unless otherwise defined, the term "substituted" means that one or more H in a group is replaced by a substituent selected from the group consisting of halogen, C 1-6 Alkyl, C 1-6 Halogenated alkyl.

[0129] As used herein, the term "reactive group" refers to a group that is susceptible to reacting with other reactive groups, thereby forming a connection between two reactive groups. In this article, reactive groups include, but are not limited to: amino (-NH2), amide (-CONH2), maleimide, bioorthogonal reactive groups, etc.

[0130] As used herein, the term "bioorthogonal group" refers to a group containing a functional group that can be used for bioorthogonal reactions, such as azide (-N3), cyclooctyne, Aldehyde, keto carbonyl, tetrazine, trans-cyclooctene, thiol, halogen, hydrazine, hydroxylamine and other groups.

[0131] As used herein, the term "amino acid residue" refers to the group formed by removing one H from the N-NH2 of an amino acid and removing the -OH from the -COOH of the C-terminus. Generally, the segment including the N-terminus and the C-terminus in the amino acid (residue) is called the main chain, and the part that determines the specific type of amino acid is called the side chain. Generally, the amino acid residue is shown as -NH-CH(R)-CO-, where R is a side chain (amino acid side chain). Unless otherwise defined, in this article, amino acids include natural amino acids or non-natural amino acids, including D-type and / or L-type amino acids. Examples of amino acids include, but are not limited to, Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), Ile (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), Val (V). Preferably, in this article, the amino acid is an amino acid selected from the following group: L-glycine (L-Gly), L-alanine (L-Ala), β-alanine (β-Ala), L-glutamic acid (L-Glu), L-aspartic acid (L-Asp), L-histidine (L-His), L-arginine (L-Arg), L-lysine (L-Lys), L-valine (L-Val), L-serine (L-Ser), L-threonine (L-Thr); in addition, when the amino acid has two or more amino groups and / or two or more carboxyl groups, the term also includes groups formed by removing one H from -NH2 and -COOH that are not on the same carbon atom, such as the divalent group -C(O)-(CH2)2-C(COOH)-NH- formed by removing one H from -NH2 of glutamic acid and non-α-position -COOH respectively.

[0132] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of about 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0133] As used herein, the term "variable" means that certain parts of the variable region in an antibody are different in sequence, which form the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments called complementary determining regions (CDRs) or hypervariable regions in the light chain and heavy chain variable regions. The more conservative parts of the variable region are called framework regions (FRs). The variable regions of natural heavy and light chains each contain four FR regions, which are generally in a β-folded configuration, connected by three CDRs that form a connecting loop, and in some cases can form a partially folded structure. The CDRs in each chain are closely together through the FR region and form the antigen binding site of the antibody together with the CDRs of the other chain. The constant region does not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody-dependent cytotoxicity of the antibody.

[0134] β-Glucan antibody drug conjugate and preparation method and application thereof

[0135] The present invention provides a β-glucan antibody drug conjugate (AGC), in particular a conjugate of an antibody for immunotherapy and β-1,3 / 1,6-glucan. The β-glucan antibody conjugate not only has a better therapeutic effect, but also improves the sensitivity of immune checkpoint antibody therapy and expands the application range of immunomodulators.

[0136] In a specific embodiment, the antibody drug conjugate has the following structure:

[0137]

[0138] Wherein, Ab, L, BG and subscript n are as described in the first aspect of the present invention.

[0139] In some embodiments, Ab is an antibody (chimeric antibody, humanized antibody, fully human antibody, nanobody).

[0140] In some embodiments, the β-glucan is a water-soluble β-1,3-glucan containing β-1,6 branches.

[0141] In some embodiments, BG is water-soluble β-1,3 / 1,6-glucan (molecular weight range 1-50 kDa).

[0142] In some embodiments, the β-glucan is a soluble β-glucan containing β-1,3 and β-1,6 glucosidic bonds in the molecule, and has a molecular weight of 1-800 kDa, preferably 1-50 kDa.

[0143] In one embodiment, the β-1,3 / 1,6-glucan is brown algae β-glucan DAG (or β-glucan DAG), preferably, the molecular weight of which is 2-6 kDa.

[0144] In one embodiment, when the β-glucan is β-glucan DAG, the β-glucan portion can be represented by the following formula:

[0145]

[0146] In one embodiment, the β-1,3 / 1,6-glucan is yeast β-glucan BYGLS2 (or β-glucan BYGLS2).

[0147] In one embodiment, when the β-glucan is β-glucan BYGLS2, the β-glucan portion is as follows:

[0148]

[0149] In some embodiments, L is a linker (linking unit) connecting Ab and BG.

[0150] In some embodiments, n represents the average number of β-glucans conjugated per antibody and n is 1-10.

[0151] In some embodiments, the immune checkpoint drug is selected from anti-PD-L1 antibodies.

[0152] In some embodiments, the antibody drug is an immune checkpoint antibody.

[0153] In some embodiments, the above-mentioned antibody is a PD-L1 antibody.

[0154] In another embodiment, the PD-L1 antibody is selected from Anti-Mouse PD-L1 (αPD-L1, 10F.9G2), and / or its molecular weight is 146671.1 Da.

[0155] In some embodiments, the above antibodies contain or are modified with various reactive groups, such as amino groups, amide groups, maleimide groups, bioorthogonal agents trans-cyclooctene, bioorthogonal agents tetrazine, bioorthogonal agents dibenzocyclooctyne, and bioorthogonal agents azide.

[0156] In some embodiments, in the above-mentioned β-1,3 / 1,6-glucan antibody conjugate, the connection site between the antibody and the linker arm L is the amino group on the lysine residue in the antibody.

[0157] In some embodiments, the use of the β-glucan antibody conjugate of the present invention includes anti-tumor effects, and the mechanism of the conjugate includes increasing the infiltration of immune cells in the tumor microenvironment and improving the tumor microenvironment.

[0158] In some embodiments, the cancer is selected from one or more of melanoma, colorectal cancer, colon cancer, lung cancer, kidney cancer, liver cancer, and breast cancer.

[0159] In some embodiments, a method for constructing a β-glucan antibody conjugate and its application are also provided, wherein the β-glucan is a soluble β-glucan containing β-1,3 and β-1,6 glucosidic bonds in the molecule, and has a molecular weight of 1-50 kDa, and is used to prepare a glucan immune checkpoint antibody conjugate, and the glucan antibody drug conjugate can be used to treat various cancers.

[0160] In some embodiments, the cancer is mouse colon cancer.

[0161] In some embodiments, the mouse colon cancer cells are MC38 and CT26 cells.

[0162] In some embodiments, the antibody can be ipilimumab, pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, cergutuzumab, ibritumomab, tiuxetan), rituximab, tositumomab, gemtuzumab, alemtuzumab, cetuximab, panitumumab, depatuxizumab, sibrotuzumab, codrituzumab, trastuzumab, patritumab, figitumumab, ganitumab, cantuzumab, bavituximab, palivizumab, bevacizumab.

[0163] In some embodiments, the β-glucan is a β-1,3-glucan containing β-1,6 branches, and has a molecular weight of 1-50 kDa.

[0164] In some embodiments, the glucan-antibody conjugate is a conjugate of β-glucan and an immune checkpoint antibody drug.

[0165] In some embodiments, the immune checkpoint antibody drug is selected from a programmed death 1 protein (PD-1) antagonist, or a PD-L1 antagonist, or a cytotoxic T lymphocyte antigen (CTLA-4) antagonist, or a lymphocyte activation gene-3 (LAG-3) antagonist, or a T cell immunoglobulin-3 (TIM-3) antagonist, or a T cell immunoglobulin and ITIM domain protein (TIGIT) antagonist.

[0166] In some embodiments, the antibodies include: chimeric antibodies, humanized antibodies, fully human antibodies, nanobodies, and the like.

[0167] In some embodiments, the immune checkpoint drug is selected from anti-PD-L1 antibodies.

[0168] In some embodiments, the use of the β-1,3 / 1,6-glucan antibody conjugate in the treatment of mouse colon cancer tumors is provided, wherein the anti-PD-L1 antibody is selected from Anti-Mouse PD-L1 (αPD-L1, 10F.9G2).

[0169] In some embodiments, the cancer is selected from one or more of melanoma, colorectal cancer, colon cancer, lung cancer, kidney cancer, liver cancer, and breast cancer.

[0170] In some embodiments, the β-1,3 / 1,6-glucan has the characteristics of good water solubility and high safety.

[0171] In some embodiments, the β-glucan is derived from brown algae or fungi; preferably, derived from brown algae.

[0172] In some embodiments, the brown algae comprises Antarctic Seagrass.

[0173] In some embodiments, the fungus comprises: yeast.

[0174] In some embodiments, the β-glucan is derived from Durvillaea antarctica, D. poha, D. chathamensis, D. potatorum, D. willana, D. amatheae.

[0175] In some embodiments, the β-glucan has a structure shown in formula (I) and / or formula (II):

[0176]

[0177] wherein n is an integer selected from 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20), and R is H and / or no more than 4 glucose residues (e.g., 1, 2, 3 or 4 glucose residues).

[0178] Preferably, R in the structure of formula (I) or formula (II) is one or more of the structures of formula (III) or formula (IV) or formula (V) or formula (VI), wherein

[0179] Formula (III): Glcβ1-;

[0180] Formula (IV): Glcβ1-3Glcβ1- or Glcβ1-6Glcβ1-;

[0181] Formula (V): Glcβ1-3Glcβ1-3Glcβ1- or Glcβ1-6Glcβ1-3Glcβ1- or

[0182] Glcβ1-3Glcβ1-6Glcβ1- or Glcβ1-6Glcβ1-6Glcβ1-;

[0183] Formula (VI): Glcβ1-3Glcβ1-3Glcβ1-3Glcβ1-or Glcβ1-6Glcβ1-3Glcβ1-3Glcβ1-or Glcβ1-3Glcβ1-6Glcβ1-3Glcβ1-or Glcβ1-3Glcβ1-3Glcβ1-6Glcβ1-or Glcβ1-6Glcβ1-6Glcβ1-3Glcβ1-or Glcβ1-6Glcβ1-3Glcβ1-or Glcβ1-6Glcβ1-3Glcβ1-or Glcβ1-6Glcβ1-3Glcβ1-6Glcβ1-or Glcβ1-3Glcβ1-6Glcβ1-6Glcβ1-or Glcβ1-6Glcβ1-6Glcβ1-or Glcβ1-6Glcβ1-6Glcβ1-6Glcβ1-.

[0184] Preferably, among the side chains of the β-1,3 / 1,6-glucan, at least 20% of the side chains have a length of 1 or 2 glucose residues.

[0185] Preferably, in the β-glucan of formula (I) or (II), at least 3 to 20 Rs are independently 1 or 2 glucose residues. Preferably, in the β-glucan of formula (I) or (II), at least 3 to 10 Rs are independently 1 or 2 glucose residues; wherein the structures of formula (III) and (IV) are as defined above.

[0186] Preferably, when the side chain (R) is 1 or 2 glucose residues in length, the side chain (R) is independently of the formula (III) and the formula (IV), wherein the formula (III) and the formula (IV) are as defined above. Preferably, when the side chain (R) is 3 or 4 glucose residues in length, the side chain (R) is independently of the formula (V) or the formula (VI), wherein the formula (V) or the formula (VI) are as defined above.

[0187] Preferably, in the side chains of the β-1,3 / 1,6-glucan (when R≠H), at least 5% of the side chains (R) are 3 or 4 glucose residues in length (preferably, 5-15%, more preferably 5-10% of the side chains are 3 or 4 glucose residues in length), and the remaining side chains (R) are 1 or 2 glucose residues in length.

[0188] In some embodiments, the β-glucan can be obtained commercially or extracted from a raw material (such as brown algae or fungi) according to methods described herein or known to those skilled in the art (such as CN105001352A, CN111690075A and CN113797221A).

[0189] In some embodiments, the β-glucan can be obtained according to the following method, which comprises the steps of:

[0190] (1) Defatting: drying and crushing the brown algae, soaking and stirring with an organic solvent to obtain defatted algae powder;

[0191] (2) water extraction: the defatted algae powder is stirred with water at a certain temperature (such as room temperature) to obtain a water extract;

[0192] (3) Classification: centrifuge the aqueous extract obtained in step (2), add 1-3 mol / L calcium chloride aqueous solution to the supernatant obtained by centrifugation; stir and centrifuge, take the supernatant and perform dialysis or ultrafiltration desalination, vacuum concentration and drying to obtain crude polysaccharide (wherein, the dialysis membrane or ultrafiltration membrane used can be selected according to the glucan to be retained);

[0193] (4) Purification: The crude polysaccharide of step (3) is dissolved in distilled water, and separated and purified by anion exchange resin using distilled water and sodium chloride aqueous solution as mobile phases, and the water elution fraction is collected, concentrated under reduced pressure, and freeze-dried to obtain the β-1,3 / 1,6-glucan.

[0194] In another preferred embodiment, the anion resin separation and purification is a strong anion resin separation and purification.

[0195] In another preferred embodiment, the anion resin separation and purification is: first separation and purification by a strong anion resin and then separation and purification by a weak anion resin; or, first separation and purification by a weak anion resin and then separation and purification by a strong anion resin. By combining a weak anion resin and a strong anion resin, unexpectedly, impurities can be removed more effectively and β-1,3 / 1,6-glucan with a longer side chain glucose residue, higher purity and a triple helix structure can be obtained. Preferably, the strong anion resin is an anion resin containing a quaternary ammonium group. Preferably, the weak anion resin is an anion resin containing a diethylaminoethyl group.

[0196] In some embodiments, the β-glucan can be used to prepare a glucan-antibody conjugate.

[0197] In some embodiments, the β-glucan antibody conjugate is used to inhibit the proliferation of mouse colon cancer cells.

[0198] In another aspect of the present invention, a method for preparing the above-mentioned conjugate is also provided, which comprises: combining β-glucan such as β-1,3 / 1,6-glucan (BG) with a linker unit (L b ) combination (L b -BG) with modified or unmodified antibodies (or antibody drugs) (Ab or Ab-L a ) is coupled to a step (wherein L aAfter coupling with β-glucan, it can form L or L b After conjugation with an antibody, it can form L or L a and L b After coupling, L) can be formed.

[0199] In some embodiments, the preparation method further comprises the step of modifying the antibody drug with a bioorthogonal agent such as trans-cyclooctene.

[0200] In some embodiments, the β-glucan antibody conjugate can be prepared by the following steps:

[0201] (1) Crosslink the antibody with a bifunctional crosslinker reagent (such as maleimide TCO-PEG with a tetrazine group at the end) n1 -NHS, wherein the number of PEG is 2-12 (i.e., n=2-12) (specifically, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12), preferably, the number of PEG is 4 (i.e., n=4)) for incubation;

[0202] (2) the antibody obtained in step (1) (i.e., Ab-L a ) and the above-mentioned combination L b -BG incubation.

[0203] In some embodiments, in step (1), the equivalent ratio of the antibody to the bifunctional cross-linking agent is 1:8-15 (specifically 1:8, 1:9, 1:10, 1:15); preferably, the reaction equivalent ratio is 1:10.

[0204] In some embodiments, the incubation temperature in step (1) is 25-30°C (specifically 25, 26, 27, 28, 29, 30°C); preferably, the incubation temperature is 28°C.

[0205] In some embodiments, the incubation time in step (1) is 0.5-3 hours (specifically 0.5, 1, 2, 3 hours); preferably, the incubation time is 0.5 hours.

[0206] In some embodiments, in step (2), the antibody and L b -The equivalent ratio of BG is 1:5-20 (such as 1:5, 1:12.5, 1:20); preferably, the equivalent ratio is 1:12.5.

[0207] In some embodiments, the incubation temperature in step (2) is 25-30°C (such as 25, 26, 27, 28, 29, 30°C); preferably, the incubation temperature is 28°C.

[0208] In some embodiments, the incubation time in step (2) is 24-48 hours (specifically, 24, 36, 48 hours); preferably, the incubation time is 48 hours.

[0209] In another embodiment of the present invention, the above preparation method further comprises a conjugate of the β-1,3 / 1,6-glucan drug and the linker unit (L b -BG) preparation steps.

[0210] In some embodiments, the conjugate of the β-1,3 / 1,6-glucan drug and the linker unit (L b -BG) can be prepared by the following steps:

[0211] (2.1) Reductive amination of β-1,3 / 1,6-glucan: An amino-modifying agent (such as a diamide linker with an amino group at the end, such as adipic acid dihydrazide ADH) is connected to the reducing end of β-glucan through a reductive amination reaction to obtain terminal amino-modified β-1,3 / 1,6-glucan;

[0212] (2.2) Binding of terminal amino-modified β-1,3 / 1,6-glucan to the linker unit: NHS active ester Tz-PEG with a tetrazine group at the end n2 -NHS ester (tetrazine-polyethylene glycol-succinimide) reacts with the terminal amino-modified β-glucan obtained in step (2.1) through an amide reaction to form an amide bond, thereby combining to obtain a β-glucan derivative with a terminal tetrazine group (L b -BG), wherein the length of the linking unit PEG is 2-12 (i.e., n2=2-12) (specifically, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12); preferably, the number of the PEG is 4 or 5, i.e., n2=4 or 5);

[0213] In some embodiments, in step (2.1), the equivalent ratio of β-1,3 / 1,6-glucan to the reducing agent (sodium cyanoborohydride) is 1:50-100 (specifically 1:50, 1:60, 1:70, 1:80, 1:90, 1:100). Preferably, the equivalent ratio is 1:100.

[0214] In some embodiments, in step (2.1), the equivalent ratio of β-1,3 / 1,6-glucan to the amino modifier to the reducing agent is 1:50-100 (specifically 1:50, 1:60, 1:70, 1:80, 1:90, 1:100). Preferably, the equivalent ratio is 1:100.

[0215] In some embodiments, the incubation temperature in step (2.1) is 40-60°C (specifically 40, 45, 50, 55, 60°C); preferably, the incubation temperature is 60°C.

[0216] In some embodiments, the incubation time in step (2.1) is 24-48 hours (specifically, such as 24, 36, or 48 hours); preferably, the incubation time is 24 hours.

[0217] In some embodiments, in step (2.2), the equivalent ratio of β-1,3 / 1,6-glucan to the linker arm L is 1:5-20 (specifically 1:5, 1:10, 1:15, 1:20). Preferably, the equivalent ratio is 1:10.

[0218] In some embodiments, the incubation temperature in step (2.2) is 40-60°C (specifically 40, 45, 50, 55, 60°C); preferably, the incubation temperature is 45°C.

[0219] In some embodiments, the incubation time in step (2.2) is 24-48 hours (specifically, 24, 36, 48 hours); preferably, the incubation time is 48 hours.

[0220] In one aspect of the present invention, a β-glucan antibody conjugate is provided for use in activating immune cells and increasing immune cell infiltration in the tumor microenvironment. Preferably, the immune cells are one or more of T cells, monocytes, neutrophils, NK cells, and dendritic cells.

[0221] In another embodiment, the β-1,3 / 1,6-glucan antibody conjugate has an anti-tumor effect.

[0222] In some embodiments, the administration route of the β-1,3 / 1,6-glucan antibody conjugates can be intraperitoneal injection or intravenous injection.

[0223] In some embodiments, the β-1,3 / 1,6-glucan antibody conjugate is administered in a coupled, combined or separate manner.

[0224] In some embodiments, the use of the β-1,3 / 1,6-glucan antibody conjugate is used to treat cancer in an individual.

[0225] Preferably, the cancer is one or more of lymphoma, melanoma, colorectal cancer, colon cancer, lung cancer (small cell lung cancer, non-small cell lung cancer), kidney cancer, gastric cancer, liver cancer, and breast cancer.

[0226] In some embodiments, the β-1,3 / 1,6-glucan antibody conjugate is used, and the therapeutic concentration of the β-1,3 / 1,6-glucan antibody conjugate is 15 mg / kg (calculated based on the PD-L1 antibody concentration).

[0227] Pharmaceutical compositions and methods of administration

[0228] As used herein, the term "the conjugate of the present invention" refers to the conjugate represented by Formula I.

[0229] Since the conjugate of the present invention has excellent anti-tumor effects, the conjugate of the present invention or a pharmaceutically acceptable salt thereof, and a composition containing the compound of the present invention as a main active ingredient can be used to treat a variety of cancers (e.g., lymphoma, melanoma, colorectal cancer, colon cancer, lung cancer, kidney cancer, gastric cancer, liver cancer, breast cancer, etc.).

[0230] The composition of the present invention comprises a safe and effective amount of the compound of the present invention or a pharmacologically acceptable salt thereof and a pharmacologically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Usually, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention / dose, more preferably, 10-500 mg of the compound of the present invention / dose. Preferably, the "one dose" is a capsule or tablet.

[0231] "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0232] There is no particular limitation on the administration of the compound or pharmaceutical composition of the present invention. Representative administrations include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

[0233] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0234] Solid dosage forms such as tablets, pills, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifiers, and the release of the active compound or compounds in such compositions can be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound can also be formed into microencapsulated form with one or more of the above-mentioned excipients.

[0235] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.

[0236] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0237] Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances, and the like.

[0238] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0239] Dosage forms for topical administration of the compounds of the invention include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required.

[0240] The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.

[0241] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage during administration is a pharmaceutically effective dosage, and for a person weighing 60 kg, the daily dosage is usually 1 to 2000 mg, preferably 20 to 500 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the health status of the patient, which are all within the skill of a skilled physician.

[0242] The main advantages of the present invention include

[0243] (1) The β-1,3 / 1,6-glucan antibody conjugate (AGC) of the present invention has a cell bridging effect, activates the innate and adaptive immune systems, and exerts a synergistic effect. For tumor cells (MC38) that are sensitive to PD-L1 treatment, the anti-tumor effect is better; for tumor cells (CT26) that are insensitive to PD-L1 treatment, AGC can also increase the sensitivity of drug treatment.

[0244] (2) The β-1,3 / 1,6-glucan PD-L1 antibody conjugate (AGC) of the present invention has a superior anti-tumor effect compared to the combined treatment of β-1,3 / 1,6-glucan and PD-L1 antibody.

[0245] (3) The β-1,3 / 1,6-glucan of the present invention is derived from brown algae in the ocean and has the characteristics of small molecular weight, good water solubility and high safety.

[0246] In general, the β-glucan backbone of the present invention is composed of glucose monomers connected by β-(1,3) glycosidic bonds, and the side chains are composed of glucose connected to the backbone by β-(1,6) glycosidic bonds; β-1,3 / 1,6-glucan is coupled with immune checkpoint antibodies to obtain a conjugate, which exerts an anti-tumor effect by improving the tumor immune microenvironment. β-glucan antibody-drug conjugates (AGC) can activate the innate and adaptive immune systems and exert a synergistic effect. Animal tumor models have shown that AGC treatment can significantly improve the anti-tumor effect of PD-L1-sensitive MC38 compared with any monotherapy and combination therapy; for PD-L1-insensitive CT26 cells, AGC can also significantly improve the effect of drug treatment.

[0247] Water-soluble β-1,3 / 1,6-glucan has good innate immune regulation and anti-tumor effects.

[0248] β-1,3 / 1,6-glucan can bind to receptors such as TLRs and Dectin-1 on the surface of immune cells such as macrophages, natural killer cells and dendritic cells, activate immune responses in the early stages of drug administration, activate immune cells, increase the release of cytokines and chemokines, and inhibit tumor growth.

[0249] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for the unrecorded specific conditions in the following examples are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0250] Example 1: Modification of the linker arm of β-glucan.

[0251]

[0252] 100 mg of β-glucan DAG (derived from Antarctic sea antlers, with a molecular weight of 2-6 kDa) was dissolved in 4 mL of DMSO: CH3COOH = 17:3 solution, and 348.4 mg of adipic acid dihydrazide ADH was added to the solution. After activation at 60°C for 30 min, 94.26 mg of sodium cyanoborohydride NaBH3CN was added, and the reaction was stopped by stirring at 60°C for 24 h. After purification by salting out and alcohol precipitation, the unreacted adipic acid dihydrazide was removed, and the product was dialyzed and freeze-dried to obtain amino-modified DAG (i.e., DAG-ADH, with a hydrogen NMR spectrum as shown in FIG. Fig.12 shown).

[0253]

[0254]

[0255] The obtained DAG-ADH was then labeled with a bioorthogonal reagent. 28 mg DAG-ADH (4 mM) and 15 mg Tz-PEG4-NHS (20 mM) were dissolved in 1.4 mL DMF, stirred at 40 °C for 24 h, and then the reaction was stopped to synthesize the linker-modified β-1,3 / 1,6-glucan (i.e., DAG-ADH-Tz, with a H NMR spectrum as shown in Formula A). Fig.12 shown).

[0256]

[0257] Example 2: Preparation of β-glucan antibody conjugate (αPD-L1-DAG).

[0258]

[0259] AGC was prepared by a two-step conjugation method. The lysine residues on the αPD-L1 antibody were modified with a bifunctional crosslinker TCO-PEG4-NHS, and the amine-reactive linker directly reacted with the lysine residues of the antibody (Anti-Mouse PD-L1 (αPD-L1, 10F.9G2)). 40 mg of the obtained αPD-L1-TCO was reacted with 18 mg of DAG-ADH-Tz at 28°C for 24 hours to finally obtain the target product αPD-L1-DAG (as shown in Formula B).

[0260]

[0261] from Figure 1 It can be seen from the figure that after the linker arm TCO-PEG4-NHS modification, the molecular weight of αPD-L1-TCO increased, proving that TCO-PEG4-NHS modification was successful. After DAG coupling, the molecular weight of αPD-L1-DAG increased significantly, proving that DAG coupling was successful. According to the protein molecule marker, a preliminary analysis of the number of DAG couplings was conducted, and 1-3 DAG molecules can be coupled to each heavy chain of αPD-L1, and 1 DAG can be coupled to each light chain.

[0262] from Figure 1 It can be seen from the SDS-PAGE electrophoresis that up to 4 DAG molecules are coupled, and the molecular weight increases by about 16000Da. The αPD-L1-DAG molecules of DAR0, DAR1, DAR2, DAR3 and DAR4 are all distributed on the SDS-PAGE electrophoresis. ImageJ software was used to analyze the proportion of different molecular weight increases to calculate the DAR value of AGC. The results are analyzed as follows: Figure 2 As shown, each αPD-L1 antibody is coupled to an average of 2.8 DAG molecules, which means that the average DAR value of αPD-L1-DAG is 2.8 (ie, n=2.8 in Formula I).

[0263] Example 3: Binding ability of β-glucan PD-L1 antibody conjugate (αPD-L1-DAG) to PD-L1 antigen (ELISA)

[0264] The enzyme-linked immunosorbent assay (ELISA) was used to verify whether the αPD-L1-DAG conjugate maintained its original affinity with the PD-L1 protein. The recombinant mouse PD-L1 protein (rmPD-L1) was coated on the ELISA plate at 4°C overnight, blocked with 5% BSA solution for 2 hours, and then αPD-L1 antibody or αPD-L1-DAG conjugate was added and incubated at room temperature for 1 hour. Goat anti-mouse IgG1 (H+L)-HRP antibody was added to the ELISA plate (1:10000 dilution), incubated at room temperature for 1 hour, and TMB was used for color development. OD650 was read by a microplate reader.

[0265] from Figure 3 It can be seen that the EC of αPD-L1-DAG affinity for PD-L1 receptor 50 =0.2065 μM, similar to naked anti-αPD-L1 (EC 50 =0.1455 μM). The experimental results show that the coupling of αPD-L1-DAG has little effect on the affinity between αPD-L1 and PD-L1.

[0266] Example 4: The ability of β-glucan PD-L1 antibody conjugate (αPD-L1-DAG) to bind to cell surface PD-L1 antigen (MC38, FACS).

[0267] The binding of αPD-L1-DAG antibody to cell surface antigens was preliminarily evaluated on MC38 (mouse colon cancer) cell lines using flow cytometry. MC38 cell suspensions were added with αPD-L1-DAG and αPD-L1 antibodies to a final concentration of 20 μg / mL, 10 μg / mL, and 1 μg / mL, respectively, and incubated on ice for 1 h. The detection antibody goat anti-mouse IgG1-FITC (1:100 dilution) was added. Flow cytometer analysis was performed.

[0268] The results showed that the MFI of MC38 cells incubated with αPD-L1-DAG was slightly different from that incubated with αPD-L1, and was significantly higher than the MFI value of the PBS-treated group (negative control group) ( Figure 4 ). This indicates that αPD-L1-DAG has not lost the ability to recognize and bind to PD-L1 on tumor cells.

[0269] Example 5: Bridging effect of β-glucan antibody conjugate (αPD-L1-DAG) on mouse MC38 colon cancer cells.

[0270] Dendritic cells from mouse bone marrow were isolated, DiD (cell membrane dye) was used to label dendritic cells, eFluor450 (cell proliferation dye) was used to label tumor cells, and finally DiD-labeled BMDCs (DiD-BMDCs) and eFluor450-labeled MC38 colon cancer tumor cells were co-cultured, and αPD-L1-DAG, αPD-L1, and PBS were added for co-incubation, and the proportion of double-positive cells in the two cell co-incubation systems was observed after different drug treatments. After the incubation, the DiD and eFluor450 staining results were detected and analyzed using a flow cytometer.

[0271] BMDCs cells and MC38 tumor cells were co-cultured. After incubation with αPD-L1-DAG, αPD-L1 and PBS at 4°C, the proportion of double-positive cells was analyzed by flow cytometry. Figure 5 It is obvious that after αPD-L1-DAG treatment, the proportion of BMDCs-MC38 double-positive cells increased, with the proportion of double-positive cells being 12.1% after αPD-L1-DAG treatment and 7.8% after αPD-L1 treatment.

[0272] Example 6: Bridging effect of β-glucan antibody conjugate (αPD-L1-DAG) on mouse B16F10 melanoma cells.

[0273] Dendritic cells from mouse bone marrow were isolated, DiD (cell membrane dye) was used to label dendritic cells, eFluor450 (cell proliferation dye) was used to label tumor cells, and finally DiD-labeled BMDCs (DiD-BMDCs) were co-incubated with eFluor450-labeled B16F10 melanoma cells, and αPD-L1-DAG, αPD-L1, and PBS were added for co-incubation, and different drug treatments were observed. The proportion of double-positive cells in the two cell co-incubation systems. The analysis method is the same as in Example 5.

[0274] BMDCs cells were co-cultured with B16F10 tumor cells. After incubation with αPD-L1-DAG, αPD-L1 and PBS at 4°C, the proportion of double-positive cells was analyzed by flow cytometry. Figure 6 It is obvious that after αPD-L1-DAG treatment, the proportion of BMDCs-B16F10 double-positive cells increased, with the proportion of double-positive cells being 23.9% after αPD-L1-DAG treatment and 6.3% after αPD-L1 treatment.

[0275] The results of Examples 5 and 6 show that in the co-incubation system, the effect of αPD-L1-DAG shortens the distance between immune cells and tumor cells, resulting in an increase in the percentage of double-positive cells of BMDCs-MC38 and BMDCs-B16F10.

[0276] Example 7: Effect of β-glucan antibody conjugate (αPD-L1-DAG) on MC38 syngeneic tumor model.

[0277] 5×10 5 A cell suspension of the mouse colon cancer cell line MC38 was injected into C57BL / 6J mice (female, 6-8 weeks old, purchased from Beijing Weitong Lihua Experimental Animal Company) under the armpit. About one week after implantation, the MC38 tumor grew to about 150 mm 3 The drug administration experiment was conducted. The mice were divided into five groups: (1) blank control group (PBS); (2) β-1,3 / 1,6-glucan group (DAG), the dose was 1 mg / kg per mouse (corresponding to the average molecular weight of DAG 4 kDa, the DAG content of 15 mg / kg dose of αPD-L1-DAG when the coupling ratio was 2.8); (3) αPD-L1 (10F.9G2, Biointron), the dose was 15 mg / kg per mouse; (4) αPD-L1-DAG, the dose was 15 mg / kg per mouse (calculated according to the amount of antibody contained); (5) β-1,3 / 1,6-glucan DAG and αPD-L1 combined treatment, the dose of combined treatment was equal to the dose of single drug treatment. Tumor volume was measured and tumor weight was recorded. After the mice were sacrificed, tumor samples were collected for flow cytometric analysis.

[0278] MC38 syngeneic tumor mice were treated with PBS, β-1,3 / 1,6-glucan (20 μg / mouse, intravenous injection, once every 3 days), αPD-L1 (10F.9G2) (300 μg / mouse, intravenous injection, once every 3 days), αPD-L1-DAG (300 μg / mouse, intravenous injection, once every 3 days), or a combination of β-1,3 / 1,6-glucan and αPD-L1 (antibody 300 μg and β-1,3 / 1,6-glucan 20 μg, intravenous injection, once every 3 days; administration started on the 7th day). Tumor volume was measured during administration. Mice were killed at the end of treatment on the 18th day to evaluate tumor weight and other indicators. Results Figure 7 As shown, compared with the PBS group, low-dose β-1,3 / 1,6-glucan DAG alone had almost no tumor inhibitory effect, and the αPD-L1 monoclonal antibody, DAG combined with αPD-L1 monoclonal antibody administration group and αPD-L1-DAG treatment group showed strong tumor growth inhibition in MC38 tumors. It was found that the tumor inhibitory activity of αPD-L1-DAG was superior to any other administration method ( Figure 7 A. Figure 7 B and Figure 7 D).

[0279] Example 8: Effects of β-glucan antibody conjugate (αPD-L1-DAG) on immune cells in the MC38 syngeneic tumor model

[0280] Take subcutaneous tumors from mice, take about 0.5g of tumor tissue, and use a mouse tumor tissue dissociation kit to obtain a single cell suspension from the tumor sample. The dissociated tumor cell suspension is subsequently processed for flow cytometry detection.

[0281] The single cell suspension was blocked with blocking buffer (10% FBS, 1:100 Mouse BD Fc Block) for 20 minutes, and incubated and stained with the corresponding immune cell surface protein antibodies (Percp-CD45, PE-CD11c, FITC-NK1.1, APC-CD3, FITC-CD8, Mouse BD Fc Block) at 4°C for 30 minutes. The proportion of immune cell populations was analyzed by flow cytometry. The specific process is to circle the cell population in the FSC / SSC quadrant, circle the single cell population by FSC-H and FSC-A, circle the corresponding immune cells by immune cell surface markers, and calculate the proportion of cells.

[0282] Analysis of immune cell subsets in tumors by flow cytometry showed that αPD-L1-DAG did not significantly increase tumor-infiltrating CD8 + However, αPD-L1-DAG significantly increased the number of tumor-infiltrating dendritic cells (CD45 + CD11c + ) and NK cells (CD45 + CD3 - NK1.1 + ) Figure 8 Specifically, compared with the PBS group and the DAG monotherapy group, the αPD-L1-DAG and αPD-L1 antibody groups can significantly increase the infiltration of NK cells in the tumor microenvironment; compared with the PBS group and the αPD-L1 antibody group, the αPD-L1-DAG and DAG monotherapy groups can lead to a significant increase in the proportion of dendritic cells in the tumor microenvironment; the increase in dendritic cells in the β-1,3 / 1,6-glucan DAG group proves that β-1,3 / 1,6-glucan can produce innate immune activation and enhance the infiltration of innate immune cells in tumor tissues.

[0283] The proportion of NK cells and dendritic cells in the αPD-L1-DAG group was higher than that in the αPD-L1 monotherapy group and the combination therapy group, which may be one of the important reasons why DAG and αPD-L1 synergistically exerted anti-tumor effects and enhanced the sensitivity of αPD-L1 anti-tumor therapy. Dendritic cells play an important role in initiating innate and adaptive immune responses.

[0284] Example 9: Effects of β-glucan antibody conjugate (αPD-L1-DAG) on immune cell infiltration, chemokines, and cytokine expression in MC38 syngeneic tumor model 24 hours after administration

[0285] Referring to Example 7, 5×10 5 A cell suspension of the mouse colon cancer cell line MC38 was injected into C57BL / 6J mice (female, 6-8 weeks old, purchased from Beijing Weitong Lihua Experimental Animal Company) under the armpit. About one week after implantation, the MC38 tumor grew to about 150 mm 3 The drug administration experiment was conducted. The mice were divided into three groups: (1) blank control group (PBS); (2) αPD-L1 (10F.9G2, Biointron), the dose was 15 mg / kg per mouse; (3) αPD-L1-DAG, the dose was 15 mg / kg per mouse (calculated according to the amount of antibody contained). The mice were killed 24 hours after intravenous injection. The tumor tissue was transferred to liquid nitrogen for quick freezing. The total RNA was extracted by TRIzol method, and sequencing (RNA-Seq) was performed after qualified quality identification. The sequencing results of each group of samples were analyzed by bioinformatics.

[0286] The abundance of several important immune cell types, including CD4 + T, CD8 + T cells, B cells, macrophages, monocytes, neutrophils, DC cells and NK cells are summarized to obtain the immune cell infiltration index. Fig. 9 As shown in the figure, the immune cell infiltration index of αPD-L1-DAG was significantly higher than that of the PBS group and the αPD-L1 monotherapy group. The clustering score calculation of the chemokine and cytokine related gene sets was performed. The calculation method was to use the expression abundance of each gene in the PBS group as the benchmark and calculate the average log2 logarithm of the fold change of each gene expression value in the related gene set in each group. The gene list of the gene set comes from the MSigDB (v2023.1.Mm) database. Fig. 9 As shown in the figure, the chemokine and cytokine-related gene set scores of αPD-L1-DAG were significantly higher than those of the PBS group and the αPD-L1 monotherapy group. The above results prove that αPD-L1-DAG produced a faster immune response 24 hours after administration, including the infiltration of immune cells, the expression of chemokines and cytokines.

[0287] Example 10: Effect of β-glucan antibody conjugates on the CT26 syngeneic tumor model.

[0288] 1×10 6 The cell suspension of mouse colon cancer cell line CT26 was injected into Balb / c mice (female, 6-8 weeks old, purchased from Beijing Weitong Lihua Experimental Animal Company) under the armpit. About two weeks after implantation, CT26 tumors grew to about 100 mm 3 The mice were divided into three groups: (1) blank control group (PBS); (2) αPD-L1 (10F.9G2), the dose was 15 mg / kg per mouse; (3) αPD-L1-DAG, the dose was 15 mg / kg per mouse (calculated according to the antibody dosage).

[0289] The experimental method was the same as that in Example 7. The mice were divided into three groups: (1) blank control group (PBS); (2) αPD-L1 (10F.9G2), at a dose of 15 mg / kg per mouse; (3) αPD-L1-DAG, at a dose of 15 mg / kg per mouse (calculated according to the dosage of the antibody); the tumor volume was evaluated and the tumor weight was recorded. PBS, αPD-L1 (10F.9G2) (300 μg / mouse, intravenous injection, once every 3 days), αPD-L1-DAG (300 μg / mouse, intravenous injection, once every 3 days) were used to evaluate the tumor volume during the administration period. The mice were killed on the 24th day to evaluate the tumor weight and other indicators. Fig.10 As shown in A, the tumors in the PBS group continued to grow, and the αPD-L1-DAG conjugate treatment improved the sensitivity of αPD-L1 monoclonal antibody treatment and had a relatively obvious tumor inhibitory effect. Fig.10 The tumor inhibition rates of each group were: αPD-L1 monoclonal antibody treatment (10.8%), αPD-L1-DAG conjugate (47.7%). Due to the rapid growth of CT26 tumors, the average volume of mice in the model group reached 2000mm after about 10 days of drug administration. 3 The average tumor volume of the blank control group (PBS) on the 9th day after administration was 1638.5 mm 3 The average tumor volume of mice in the αPD-L1 monoclonal antibody treatment group was 1380.1 mm 3 The average tumor volume of mice in the αPD-L1-DAG treatment group was 890 mm 3 ( Fig.10 A). The average tumor weight of the blank control group (PBS) on day 9 was 1.134 g, the average tumor weight of the mice in the αPD-L1 monoclonal antibody treatment group was 1.011 g, and the average tumor weight of the mice in the αPD-L1-DAG treatment group was 0.5932 g ( Fig.10B). On the 9th day of administration, the αPD-L1 monoclonal antibody treatment group still could not inhibit the growth of CT26 tumors, but αPD-L1-DAG could significantly improve the sensitivity of CT26 tumor treatment ( Fig.10 A and Fig.10 C).

[0290] The mechanism of good anti-tumor activity of αPD-L1-DAG conjugate may be:

[0291] 1) αPD-L1-DAG binds to the PD-L1 receptor on the surface of MC38 tumor cells, acting as a PD-L1 / PD-1 signaling pathway blocker, relieving the immunosuppressive state of T cells and exerting an adaptive immune anti-tumor effect;

[0292] 2) αPD-L1-DAG activates immune response in the early stage of drug administration, increases the infiltration of dendritic cells and NK cells in tumor tissue, and has an innate immune regulation effect. The above research results suggest that innate immune cells (dendritic cells and NK cells) and adaptive immune cells (CD8 + The simultaneous activation of αPD-L1-DAG and αPD-L1-DAG may be the mechanism of action of αPD-L1-DAG's anti-tumor activity in vivo. Since immune cells play an important role in a variety of tumor cell types, β-glucan antibody conjugates will play a role in a variety of tumor cells (for example, lymphoma, melanoma, colorectal cancer, colon cancer, lung cancer, kidney cancer, gastric cancer, liver cancer, breast cancer, etc.).

[0293] In addition, immune cells also play a regulatory role in tumor metastasis and occurrence. Therefore, β-glucan PD-L1 antibody conjugates may inhibit the metastasis of tumor cells.

[0294] Example 11: Other β-glucan antibody conjugates

[0295] Other β-glucan antibody conjugates synthesized in this article and their in vivo anti-tumor effects are shown in the following table:

[0296] Conjugate code β-Glucan Antibody Coupling method Anti-tumor effect Con-1 αPD-L1-DAG DAG αPD-L1 Antibody two-step method most Con-S1 αPD-1-DAG DAG αPD-1 Antibody two-step method Better Con-S2 Cetu-DAG DAG Cetuximab Antibody two-step method good Con-S3 BYGLS2-PD-L1 BYGLS2 αPD-L1 Antibody two-step method Better Con-S4 αPD-L1-DAG-SMCC DAG αPD-L1 Antibody One-Step Better

[0297] Example 11a: Effect of β-glucan DAG-PD-1 antibody conjugate (αPD-1-DAG) on MC38 syngeneic tumor model.

[0298] Referring to Examples 1 and 2, the difference is that the αPD-L1 antibody used in Example 2 is replaced with a mouse PD-1 antibody (RMP1-14, Biointron), and a conjugate of β-1,3 / 1,6-glucan DAG and mouse PD-1 antibody (RMP1-14, Biointron) is prepared, and the average coupling ratio is 3.5 (i.e., the average number of glucans coupled to the antibody, n=3.5).

[0299] 5×10 5 A cell suspension of the mouse colon cancer cell line MC38 was injected into the axilla of C57BL / 6J mice (female, 6-8 weeks old, purchased from Beijing Weitong Lihua Experimental Animal Company). About one week after implantation, the MC38 tumor grew to about 100 mm 3 The drug administration experiment was conducted. The mice were divided into four groups: (1) blank control group (PBS); (2) β-1,3 / 1,6-glucan group (DAG), the dose was 1.1 mg / kg per mouse (corresponding to the average molecular weight of DAG 4 kDa, coupling ratio 3.5, and DAG content of αPD-1-DAG at a dose of 15 mg / kg); (3) αPD-1 (RMP1-14, Biointron), the dose was 15 mg / kg per mouse; (4) αPD-1-DAG, the dose was 15 mg / kg per mouse (calculated according to the amount of antibody contained). The tumor volume was measured and the tumor weight was recorded. Intravenous injection was performed once every 3 days; drug administration began on the 7th day, and the tumor volume was measured during the drug administration. The mice were killed at the end of treatment on the 18th day to evaluate indicators such as tumor weight.

[0300] The results are as follows Fig.11 As shown in (a), compared with the PBS group, low-dose β-1,3 / 1,6-glucan DAG alone had almost no tumor inhibitory effect, and the αPD-1-DAG treatment group showed a certain tumor growth inhibitory effect in MC38 tumors.

[0301] Example 11b: Effect of β-glucan DAG-cetuximab conjugate (Cetu-DAG) on HT29 xenograft tumor model.

[0302] Referring to Examples 1 and 2, the difference is that the αPD-L1 antibody used in Example 2 is replaced with cetuximab (ERBITUX) to prepare a conjugate of β-1,3 / 1,6-glucan DAG and cetuximab, and the average conjugation ratio is 1.0 (n=1.0).

[0303] 5×10 5 The cell suspension of the human colon cancer cell line HT29 was injected into Balb / c nude mice (female, 6-8 weeks old, purchased from Beijing Weitonglihua Experimental Animal Company) under the armpit. About one week after implantation, the HT29 tumor grew to about 100 mm 3The drug administration experiment was conducted. Nude mice were divided into four groups: (1) blank control group (PBS); (2) β-1,3 / 1,6-glucan group (DAG), the dose was 0.9 mg / kg per mouse (corresponding to the average molecular weight of DAG 4 kDa, the DAG content of 18 mg / kg dose of Cetu-DAG when the coupling ratio was 1.0); (3) Cetu (Cetuximab, ERBITUX), the dose was 18 mg / kg per mouse; (4) Cetu-DAG, the dose was 18 mg / kg per mouse (calculated according to the amount of antibody contained). Intravenous injection, once every 3 days; drug administration started on the 7th day), the tumor volume was measured during the drug administration, and the mice were killed at the end of treatment on the 21st day to evaluate indicators such as tumor weight.

[0304] The results are as follows Fig.11 As shown in (b), compared with the PBS group, low-dose β-1,3 / 1,6-glucan DAG alone and Cetu monoclonal antibody had no significant inhibitory effect on tumor growth in HT29 tumors, and the Cetu-DAG administration group had a certain tumor inhibition activity.

[0305] Example 11c: Effect of β-glucan BYGLS2-PD-L1 antibody conjugate (αPD-L1-BYGLS2) on the MC38 syngeneic tumor model.

[0306] In this example, brewer's yeast is used as a raw material to extract and prepare water-soluble yeast β-glucan BYGLS2 (the preparation method can be found in CN111690075A), whose main chain structure is β-1,3-glucan, with a branched structure at the C6 position of the glucose residue, and a molecular weight of about 280 kDa. Its structure is shown in the following formula:

[0307]

[0308] Referring to Examples 1 and 2, a conjugate of β-glucan DAG and mouse PD-L1 antibody (10F.9G2, Biointron) αPD-L1-BYGLS2 was prepared with an average conjugation ratio of 1.0.

[0309]

[0310] 5×10 5 A cell suspension of the mouse colon cancer cell line MC38 was injected into the axilla of C57BL / 6J mice (female, 6-8 weeks old, purchased from Beijing Weitong Lihua Experimental Animal Company). About one week after implantation, the MC38 tumor grew to about 100 mm 3The drug administration experiment was conducted. The mice were divided into four groups: (1) blank control group (PBS); (2) β-1,3 / 1,6-glucan BYGLS2 group (BYGLS2), the dose was 15 mg / kg per mouse (corresponding to the BYGLS2 average molecular weight of 280 kDa, the BYGLS2 content of 15 mg / kg dose of αPD-L1-BYGLS2 when the coupling ratio was 1.0); (3) αPD-L1 (10F.9G2, Biointron), the dose was 15 mg / kg per mouse; (4) αPD-L1-BYGLS2, the dose was 15 mg / kg per mouse (calculated according to the amount of antibody contained). The tumor volume was measured and the tumor weight was recorded. Intravenous injection, once every 3 days; drug administration started on the 7th day), the tumor volume was measured during the drug administration, and the mice were killed at the end of treatment on the 18th day to evaluate indicators such as tumor weight.

[0311] The results are as follows Fig.11 As shown in c, compared with the PBS group, BYGLS2 alone had no significant tumor inhibitory effect, and the αPD-L1-BYGLS2 treatment group showed significant tumor growth inhibition in MC38 tumors.

[0312] Example 11(d): Construction of DAG and PD-L1 antibody conjugate (αPD-L1-DAG-SMCC) by one-step antibody conjugation method and its effect on MC38 syngeneic tumor model.

[0313] In this example, adipic acid dihydrazide (ADH) and sulfosuccinimide 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) were reacted with β-1,3 / 1,6-glucan DAG to construct the L-BG unit:

[0314]

[0315] The PD-L1 antibody (10F.9G2, Biointron) was reduced with tri(2-carbonylethyl)phosphine hydrochloride (TCEP), and then the L-BG unit constructed above was directly coupled with the cysteine ​​group on the antibody to construct a DAG and PD-L1 antibody conjugate (αPD-L1-DAG-SMCC) with an average coupling ratio of 1.5 (n=1.5).

[0316]

[0317] 5×10 5 A cell suspension of the mouse colon cancer cell line MC38 was injected into C57BL / 6J mice (female, 6-8 weeks old, purchased from Beijing Weitong Lihua Experimental Animal Company) under the armpit. About one week after implantation, the MC38 tumor grew to about 150 mm 3The mice were divided into four groups: (1) blank control group (PBS); (2) β-1,3 / 1,6-glucan group (DAG), the dose was 0.6 mg / kg per mouse (corresponding to the average molecular weight of DAG 4 kDa, coupling ratio 1.5, and DAG content of αPD-1-DAG at a dose of 15 mg / kg); (3) αPD-L1 (10F.9G2, Biointron), the dose was 15 mg / kg per mouse; (4)

[0318] αPD-L1-DAG-SMCC, the dose is 15 mg / kg per mouse (calculated according to the amount of antibody contained). Measure the tumor volume and record the tumor weight. Intravenous injection, once every 3 days; start administration on the 7th day, measure the tumor volume during administration, end treatment on the 15th day and kill the mice to evaluate indicators such as tumor weight.

[0319] The results are as follows Fig.11 As shown in (d), the αPD-1-DAG-SMCC treatment group showed significant tumor growth inhibition in MC38 tumors compared with the PBS group.

[0320] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that the scope of the present invention is defined by the claims attached to the present application.

Claims

1. A conjugate or a pharmaceutically acceptable salt thereof, characterized in that: The conjugate is shown in formula (I) in, Ab is an antibody portion, and the antibody is an antibody targeting an immune checkpoint or an antibody targeting a receptor for signal transduction; BG is the β-glucan part; L is a linker that connects the antibody and β-glucan; and The subscript n is 1 to 10.

2. The conjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The antibody targeting the immune checkpoint is selected from the group consisting of a PD-L1 antagonist, a programmed death 1 protein (PD-1) antagonist, a cytotoxic T lymphocyte antigen (CTLA-4) antagonist, a lymphocyte activation gene-3 (LAG-3) antagonist, a T cell immunoglobulin-3 (TIM-3) antagonist, a T cell immunoglobulin and ITIM domain protein (TIGIT) antagonist, or a combination thereof; and The antibody targeting the signal transduction receptor is an epidermal growth factor receptor antagonist (EGFR antagonist).

3. The conjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The antibody is an antibody targeting an immune checkpoint, and the antibody targeting an immune checkpoint is selected from the following group: an anti-PD-L1 antibody, an anti-PD-1 antibody, or a combination thereof.

4. The conjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The β-glucan is derived from brown algae or fungi.

5. The conjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: Ab represents anti-PD-L1 antibody or anti-PD-1 antibody; and BG is a β-glucan portion, the β-glucan being derived from Antarctic sea antlers.

6. The conjugate or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The conjugate is shown in formula III-a or III-b Wherein, Ab, BG and n are as defined in claim 1; s1 is an integer from 1 to 20; s2 is an integer from 1 to 10; s3 is an integer from 1 to 15; and s4 is an integer from 0 to 10.

7. The conjugate or a pharmaceutically acceptable salt thereof according to claim 6, characterized in that: Ab stands for anti-PD-L1 antibody or anti-PD-1 antibody; BG is a β-glucan part, and the β-glucan is derived from Antarctic sea antlers; s1=1, 2, 3, 4, 5, 6, 7 or 8; s2=3, 4, 5, 6 or 7; s3=2, 3, 4, 5 or 6; s4 = 1 or 2; and n is 1 to 4.

8. A pharmaceutical composition comprising (a) the conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, and (b) a pharmaceutically acceptable carrier or excipient.

9. Use of the conjugate or a pharmaceutically acceptable salt thereof as claimed in claim 1, or a pharmaceutical composition comprising the conjugate or a pharmaceutically acceptable salt thereof, in the preparation of an anti-tumor or cancer treating drug.

10. The use according to claim 9, characterized in that The cancer is selected from the group consisting of melanoma, colorectal cancer, colon cancer, lung cancer, kidney cancer, liver cancer, breast cancer, or a combination thereof.

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