Conjugates of anti-CTLA-4 antibodies activated in the tumor microenvironment and uses thereof

Abstract

The present invention provides a conjugate of an anti-CTLA-4 antibody activated in the tumor microenvironment and its use. The conjugate of the present invention is R1-R2-R3-L-R4-S-cys-R5, where L, R4, S, cys, and R5 each represent structures such as different functional groups, spacer arms, chemical bonds, and CTLA-4 antibodies. The conjugate of the present invention is a dual activation conjugate, which can effectively enhance the target efficacy of the anti-CTLA-4 antibody, overcome the drug resistance of the antibody, and reduce toxicity.

Description

Technical Field

[0001] The present invention relates to a conjugate of an anti-CTLA-4 antibody activated in a tumor microenvironment and its use.

Background Art

[0002] Cancer immunotherapy is one of the epoch-making advances in the field of cancer treatment in recent years, and in some patients, cancer immunotherapy enables long-term survival. Cancer immunotherapies include cancer immune checkpoint antibodies, bispecific T cell engagers (bsTCE), chimeric antigen receptor cells (CAR-T), oncolytic viruses, and the like. Among them, the CTLA-4 antibody is the first immune checkpoint inhibitor antibody approved by the FDA. The PD-1 antibody is another immune checkpoint inhibitor with remarkable clinical therapeutic effects. CTLA-4 antibodies, PD-1 antibodies, and combination therapies of both have obtained very excellent therapeutic effects in clinical trials including cancers such as melanoma, non-small cell lung cancer, lymphoma, and liver cancer, and some treatment methods have already become first-line therapies.

[0003] Immune checkpoint antibodies often cause T cells to be non-specifically activated in non-tumor target organs in order to relieve the inhibitory signaling pathway of T cells, and as a result, immune-related adverse events (irAEs) are caused. On the other hand, the response rate of single-agent treatment with immune checkpoint antibodies is usually limited to about 20-30% in many cases. It is necessary to increase the response rate of patients by adopting combination immunotherapy such as PD-1+CTLA-4 dual-target immunotherapy. However, this combination also significantly enhances the irAEs effect. For example, in a clinical study of treating malignant melanoma by combining CTLA-4 and PD-1, the overall response rate is 58%, which is significantly improved compared with CTLA-4 alone (19%) or PD-1 alone (44%). However, at the same time, with the improvement of the treatment effect, more serious treatment-related adverse events (TRAEs) are also observed clinically. Among them, grade 3 to grade 4 TRAEs increased to 55%, and the incidence rate was significantly higher than that of CTLA-4 alone (27%) or PD-1 alone (16%). Common irAEs include hypophysitis, hepatitis, colitis, rash, diarrhea, etc. These serious irAEs have caused the death of the subjects.

[0004] CTLA-4 is a receptor that suppresses the surface expression of T cells. Its mechanism is to competitively bind B7.1 (CD80) and B7.2 (CD28) with CD28. After T cells receive the antigen presented by MHC, they will not cause excessive activation. When this inhibitory signal is relieved by a CTLA-4 antibody, such as Ipilimumab, the activation and proliferation of T cells after being stimulated by the MHC-antigen complex will increase, and the tumor immune effect mediated by T cells will be improved. At the same time, recent studies have shown that CTLA-4 is highly expressed on the surface of Treg cells, and a CTLA-4 antibody with Fc effector function, such as Ipilimumab, can remove the Treg cells inside the tumor through the ADCC effect mediated by IgG1 Fc, relieve the microenvironment of tumor suppression, and play a role in enhancing anti-tumor immunity.

[0005] As described above, any effect, whether based on the release of T cell activation inhibitory signals or on the killing of Treg cells by the ADCC effect, promotes autoimmune reactions in normal organs and leads to the occurrence of irAEs. Therefore, clinically, in order to limit the immune-enhancing effect to the tumor interior and avoid autoimmune toxicity to normal tissues, it is necessary to develop a safer anti-CTLA-4 antibody.

[0006] Contents of the Invention Based on the above technical problems, the present invention provides a conjugate of a tumor microenvironment-activated anti-CTLA-4 antibody having the following structure: R1-R2-R3-L-R4-S-cys-R5 However, R5 represents an anti-CTLA-4 antibody in which one or more amino acid residues are mutated to cysteine; cys represents the cysteine residue of R5; S represents the S atom in the cysteine residue; R1 is a functional group that blocks the binding of R5 to its antigen, ligand or receptor; R2 is a spacer arm that can be hydrolytically cleaved by one or more proteases in the pathological microenvironment; R3 is a spacer arm that provides space for protease cleavage; L is a chemical bond that is activated under acidic conditions in the pathological microenvironment; R4 is a group that covalently binds to R5 via the sulfur atom on the cysteine in R5, and can restore, maintain or improve the binding ability of R5 to its antigen, ligand and receptor after the cleavage of R1-R2-R3-L-.

[0007] The present invention provides a pharmaceutical composition comprising the conjugate described herein.

[0008] The present invention provides a method for treating or preventing a tumor, which comprises providing a therapeutically or prophylactically effective amount of the conjugate described in any one of the embodiments herein to a subject in need thereof.

[0009] The present invention further provides the use of the conjugate described herein in the preparation of a drug for treating or preventing tumors.

[0010] The present invention provides the conjugate described in any one of the embodiments herein for use in therapeutic or prophylactic treatment.

Brief Description of the Drawings

[0011]

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[0012] Specific Embodiments Within the scope disclosed by the present invention, it should be understood that each related technical feature can be combined with the technical features described later, including the technical features listed in the examples, to form preferred technical means for solving technical problems. Furthermore, the content disclosed by the present invention is not limited to the specific examples described, and therefore, there may be one or more changes. It should be understood that all technical terms and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs, unless specifically defined otherwise. Any other methods and materials similar or equivalent to those described in this specification can also be used in the practice or testing of the present invention, but the methods and materials described in this specification are preferred. All publications mentioned in this specification are incorporated herein by reference, disclosing and explaining the methods and / or materials related to the cited publications.

[0013] As used in this specification and the appended claims, it should be noted that the singular forms "a", "one", "the", and "this" include plural referents unless the context clearly dictates otherwise. It should also be noted that claims can be drafted to exclude any optional elements. Accordingly, this statement is intended to serve as a basis for using exclusive terms such as "solely", "only", etc. or "negative" limitations in the description of the elements of the claims.

[0014] One of the objectives of the present invention is to provide a modified anti-CTLA-4 antibody conjugate that is activated only in the pathological microenvironment (such as the tumor microenvironment or inflammatory sites), and by releasing an anti-CTLA-4 antibody with a comparable or improved binding affinity to its ligand, it improves the target efficacy of biomolecules, overcomes drug resistance of drugs, and reduces toxicity.

[0015] In the present invention, the modified anti-CTLA-4 antibody is a conjugate that includes an anti-CTLA-4 antibody and a functional moiety that covalently binds to the antibody, namely R1-R2-R3-L-R4.

[0016] As used in the disclosure of this specification, "antibody" is used in the broadest sense and includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments as long as they exhibit the desired biological activity (Miller et al., (2003), Jour. of Immunology, 170:4854-4861). In the context and drawings, the abbreviation for antibody is replaced by "Ab".

[0017] The basic antibody structural unit comprises a tetramer consisting of two pairs of the same polypeptide chains, with each pair having one light chain and one heavy chain. The N-terminal portion of each chain contains a variable region of approximately 100-110 or more amino acids that is mainly responsible for antigen recognition. The C-terminal portion of each chain defines a constant region that is mainly responsible for effector functions. The heavy chain variable region (VH) and the light chain variable region (VL) each contain three complementarity-determining regions (CDRs), including HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3. These six CDRs form the antigen-binding site of the antibody. The remaining amino acids of the variable region are relatively conserved and are called framework regions (FR). VH and VL each contain four framework regions, called FR1, FR2, FR3 and FR4 respectively.

[0018] Antibodies can be derived from mice, humans or other substances and may be humanized antibodies or chimeric antibodies. The immunoglobulins disclosed in the present invention may be of any type (e.g., IgG, IgE, IgM, IgD and IgA), category (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), or subcategory of immunoglobulin molecules. The immunoglobulins may be derived from any species, e.g., humans, mice or rabbits.

[0019] Antibody fragments contain a part of the full length of an antibody and usually contain the antigen-binding region or variable region of the antibody. Preferably, the antibody fragment is a functional fragment, i.e., one that retains the antigen-binding ability of the complete antibody, i.e., the antibody fragment is an antigen-binding fragment of the antibody. Examples of antibody fragments or functional fragments include Fab, Fab’, F(ab’)2 and Fv fragments; diabodies; linear antibodies; and single-chain antibody molecules (scFv); etc.

[0020] In the present invention, one or more (for example, 5 or less or 3 or less) amino acids at appropriate positions in the amino acid sequence of the anti-CTLA-4 antibody are mutated to cysteine, and then covalently bonded to the functional portion (R1-R2-R3-L-R4) of the present invention via the mercapto group of cysteine. For example, one or two amino acids of interest among the anti-CTLA-4 antibodies of interest are mutated to cysteine and bonded to the functional portion. The position of the mutation may be in the complementarity-determining region or non-complementarity-determining region of the variable region. Preferably, the mutation is a substitution-type mutation. More preferably, the mutation occurs in the light chain variable region of the anti-CTLA-4 antibody. Usually, mutants can be prepared and the binding activity to the corresponding antigen can be measured. When the mutant retains 70% or more, preferably 80% or more, more preferably 90% or more of the binding activity compared to the wild-type antibody, the amino acid residue at the mutation site is considered to be mutated to cysteine and can covalently bond to the functional portion. Alternatively, in certain embodiments, when the conjugate produced by linking the mutant to R4 described herein retains 80% or more, preferably 90% or more, more preferably 95% or more of the binding activity, the amino acid residue at that position is considered to be able to covalently bond to the functional portion as cysteine.

[0021] All kinds of anti-CTLA-4 antibodies can be used in the present invention. A preferred anti-CTLA-4 antibody is Ipilimumab, and its exemplary heavy chain and light chain amino acid sequences are shown in SEQ ID NO: 1 and 2, respectively.

[0022] Generally, one or more, more preferably one or more, still more preferably one or more from G, A, S, T, L, I, F, E, K, D and Y, etc. in the CDRs of the light chain variable region of an anti-CTLA-4 antibody may be mutated to cysteine. In some embodiments, one or more, more preferably one or more, still more preferably one or more from G, A, S, T, L, I, K and Y, etc. in the CDRs of the heavy chain variable region of an anti-CTLA-4 antibody may be mutated to cysteine. When the mutation occurs in the non-CDR region of the anti-CTLA-4 antibody light or heavy chain variable region, one or more, preferably one or more from G, A, S, T, L, I, F, E, K, D and Y, etc. in the non-CDR of the light or heavy chain variable region may be mutated to cysteine, more preferably one or more from G, A, S, T, K, I, Y and L, still more preferably one or more from G, A, T, Y and S. In some embodiments, one or more of the S, T, L, I, F, E, K, D, N, Q, R and Y residues, etc. in the non-complementary determining region (e.g., FR1, FR2 or FR3) of the anti-CTLA-4 light or heavy chain variable region may be mutated to cysteine. In some embodiments, substitution mutations may be introduced into one or more of the following conserved sites: Gln3, Ser7, Ser25, Glu46, Thr69 and Asp73 in the non-complementary determining region (FR1, FR2 or FR3) of VH, and Thr5, Tyr50, Arg62, Ser64, Ser66, Ser68, Thr73, Thr75, Ser77 and Asp83 in the non-complementary determining region (FR1, FR2 or FR3) of VL. The above position numbers refer to SEQ ID NO: 1 and 2.

[0023] In a preferred embodiment, the mutation positions on the heavy chain of the anti-CTLA-4 antibody Ipilimumab are selected from Gln3, Arg19, Leu20, Ser25, Gly26, Phe27, Thr28, Phe29, Ser30, Ser31, Tyr32, Thr33, Met34, His35, Gly44, Phe50, Ile51, Ser52, Tyr53, Asp54, Gly55, Asn56, Asn57, Lys58, Tyr59, Tyr60, Ala61, Asp62, Ser63, Lys65, Gly66, Thr69, Ser71, Arg72, Asp73, Asn74, Ser75, Lys76, Asn77, Thr99, Gly100, Trp101, Leu102, Gly103, Pro104, Leu105, Asp106 and Tyr107; preferably, they are selected from Ser30, Ser31, Tyr32, Thr33, Ile51, Asp54, Gly55, Asn56, Lys58, Tyr59, Tyr60, Ala61, Asp62, Ser63, Lys65, Gly66, Gly100, Trp101, Leu102, Gly103, Pro104, Leu105, Asp106 and Tyr107.

[0024] In a preferred embodiment, the mutation positions in the light chain of the anti-CTLA-4 antibody Ipilimumab are selected from Gln6, Arg24, Ala25, Ser26, Gln27, Ser28, Val29, Gly30, Ser31, Ser32, Tyr33, Leu34, Ala35, Tyr37, Ile49, Tyr50, Gly51, Ala52, Phe53, Ser54, Arg55, Ala56, Thr57, Gly58, Ile59, Pro60, Asp61, Arg62, Ser68, Gly69, Thr70, Gln90, Gln91, Tyr92, Gly93, Ser94, Ser95, Pro96, Trp97, Thr98, Phe99 and Gly100; preferably, they are selected from Ala25, Ser26, Ser28, Gly30, Ser31, Ser32, Leu34, Ala35, Gly51, Ala52, Ser54, Ala56, Thr57, Gly58, Ile59, Gly93, Ser94, Ser95, Thr98 and Gly100.

[0025] The above position numbers refer to SEQ ID NO: 1 and 2.

[0026] (CN201880072546.4) It has been found that mutants obtained by mutating the amino acid residues of the FR and CDR regions of the anti-CTLA-4 antibody light chain variable region and heavy chain variable region still maintain high binding affinity (more than 60% of the wild-type antibody binding affinity). All content regarding the CTLA-4 mutants disclosed in this application is incorporated herein by reference.

[0027] Mutagenesis, transfection, expression, and purification of biomolecules can be performed by methods known in the art. For example, nucleic acids with anti-CTLA-4 antibodies mutated at selected positions can be directly synthesized, and nucleic acid molecules of different fragments obtained by enzymatic digestion can be ligated to an expression vector, and the expression vector can be transformed into bacterial or eukaryotic host cells. Biomolecules containing cysteine mutations can be obtained by recombination in host cells.

[0028] The bacteria or eukaryotic host cells suitable for the present invention may be host cells commonly used in the art, including but not limited to bacteria, yeast, and mammalian cells. Useful eukaryotic host cells include CHO cells, HEK293T cells, or Pichia pastoris.

[0029] The expression vectors suitable for the present invention may be viral expression vectors known in the art, including but not limited to vectors based on baculovirus, simian virus (SV40), retrovirus, and vaccinia virus. Expression vectors containing appropriate control elements and selection markers can be used for the production of mammalian cell lines that stably express mutants. For example, the GS eukaryotic expression system (Lonza), the DHFR eukaryotic expression system (Invitrogen), and the Pichia pastoris expression system (Invitrogen) can be used for expression and preparation.

[0030] The biomolecules of the present invention can be purified by separation methods known in the art. These methods include, but are not limited to, affinity chromatography, DEAE ion exchange columns, gel filtration, and hydroxyapatite chromatography. For example, Protein A columns or G columns can be used to separate antibody molecules from cell culture supernatants or cytoplasmic extracts. In some embodiments, the biomolecules can be "engineered" to include an amino acid sequence that can capture the biomolecule on an affinity substrate. For example, markers can be used to facilitate the purification of polypeptides. Suitable markers include, but are not limited to, c-myc, hemagonium, poly-His (e.g., 6His), and Flag TM (Kodak). Such markers can be inserted at any position within a polypeptide that includes a carboxyl group terminus or an amino group terminus. Also, the biomolecule conjugates of the present invention can be purified by immunoaffinity chromatography.

[0031] The functional group moiety suitable for the present invention can be represented by the formula R1-R2-R3-L-R4. Among the functional groups, R1, R2, and R3 are linked by any suitable linking method and include, but are not limited to, amide bond, ester bond, urethane bond, urea bond, or hydrazone bond. R3 and R4 are connected via L, and L is a hydrazone bond. In the present invention, the amide bond can be represented by “-CO-NH-”, the ester bond by “-C(O)O-”, the urethane bond by “-NH-C(O)-O-”, the urea bond by “-NH-CO-NH-)”, and the hydrazone by “-C(R’)=N-NH-”, provided that R’ is H or a C1-4 alkyl group. In this specification, unless otherwise specified, R1, R2, R3, L, R4, and R5 are covalently bonded to each other. Also, the following should be understood: when R1, R2, R3, L, R4, and R5 are part of a molecule, they refer to groups; when they are raw materials for production, they refer to compound molecules. In this case, at the bonding position (i.e., the position indicated by the following wavy line), usually, H or OH or other corresponding groups well-known in the art are bonded so as to satisfy the valence bond theory, and at the same time, they can also react with other parts to form the bonds described in this specification, including, but not limited to, the above-mentioned amide bond, ester bond, urethane bond, urea bond, or hydrazone bond.

[0032] In this specification, R1 is a protecting group for the biomolecule R5. It is selected from any group that can prevent the binding of the biomolecule to its antigen, ligand, or receptor and can prevent the biomolecule from being interfered with by other molecules. For example, it can prevent the binding of the biomolecule to its ligand or receptor before the biomolecule reaches a pathological microenvironment such as a tumor or an inflammatory microenvironment. Suitable R1 is selected from the following: NR a R b -R-CO-, C 1-4 alkoxy-(C 1-4 alkoxy) n -carbonyl-C 1-4 alkylene-carbonyl-, TIFF2025520468000001.tif59170

[0033] However, each R is independently C 1-4 an alkyl group; each n is independently an integer in the range of 1 to 30000, such as 1 - 15000, 1 - 5000, 1 - 2000, 1 - 150, 1 - 50, 1 - 20 or 3 - 12; polyethylene glycol or peg m is polyethylene glycol with a molecular weight of 44 to 132000, such as polyethylene glycol with a molecular weight of 1000 to 50000 or 10000 to 30000; m represents the molecular weight of polyethylene glycol; the wavy line indicates the position where R1 is linked to R2; R a is C 1-4 alkyl - O - [(CH2) q - O] n -(CH2) q -; R b is C 1-4 alkyl - O - [(CH2) q - O] n -(CH2) q - C(O)-; q is an integer from 1 to 4. Preferably, the molecular weight of R1 is 20 kD or less.

[0034] In some embodiments, the NR a R b - R - CO - is TIFF2025520468000002.tif30170

[0035] H Preferably, each n is independently an integer from 1 - 1000.

[0036] In some embodiments, the C 1-4 alkoxy - (C 1-4 alkoxy) n - carbonyl - C 1-4 alkylene - carbonyl - is TIFF2025520468000003.tif24170

[0037] Preferably, n is an integer from 1 - 1000.

[0038] In some embodiments, the R1 group is selected from the following: TIFF2025520468000004.tif210170

[0039] Generally, when the position of the mutation is within the functional domain of the biomolecule, for example, within the CDR of an antibody, the molecular weight of R1 is not particularly limited, and R1 may have a relatively low molecular weight. When the position of the mutation is outside the functional domain of the biomolecule, for example, in the non-CDR of an antibody, R1 is selected such that the molecular weight of R1-R2-R3-L-R4 is greater than 200, preferably greater than 500, more preferably greater than 1000, and it is preferable that the molecular weight of the conjugate of the biomolecule is 5000 or more, preferably 8000 or more, more preferably 10000 or more. Thereby, it is possible to better prevent the biomolecule from binding to its ligand or receptor before reaching the pathological microenvironment.

[0040] In the present invention, R2 is a spacer arm that is hydrolytically activated by one or more proteolytic enzymes, proteases or peptidases in the pathological microenvironment. In the present invention, the proteolytic enzyme, protease or peptidase may be any proteolytic enzyme, protease or peptidase present in the pathological microenvironment. For example, the protease may be cysteine protease, aspartic protease, aspartic acid protease, glutamic acid protease, threonine protease, gelatinase, metalloprotease or aspartic peptidase; preferably, R2 is a peptide cleavable by at least one selected from the following enzymes: asparaginyl endopeptidase, granzyme, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, kallikrein, hK1, hK10, hK15, plasmin, collagenase, type IV collagenase, stellate collagenase, factor Xa, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtilisin-like protease, actinase, bromelain, calpain, caspase, caspase-3, defensive caspase, papain, HIV-1 protease, HSV protease, CMV protease, chymotrypsin, pepsin, lipoprotein lipase, fibrinoridine, nepenthesin, metalloexopeptidase, metalloendopeptidase, matrix metalloprotease (MMP), MMP1, MMP2, MMP3, MMP8, MMP9, MMP10, MMP11, MMP13, MMP14, ADAM10, ADAM12, urokinase-type plasminogen activator (uPA), neurokinase, prostate specific antigen (PSA, hK3), interleukin 1β converting enzyme, thrombin, FAP (FAP-a), meprin, dipeptidyl peptidase and dipeptidyl peptidase IV (DPPIV / CD26). In a preferred embodiment, the disclosure of the present invention relates in particular to Legumain, which is mainly expressed and secreted by tumor cells in the tumor microenvironment. Due to the expression of Legumain, tumor-associated macrophages (M2 type) are also different from monocytes and inflammatory macrophages (M1 type).In the present invention, the polypeptide is a substrate of a proteolytic enzyme, is recognized by the proteolytic enzyme, and is cleaved by the proteolytic enzyme.

[0041] The R2 group of the present invention is represented by -R2a-, -R2b-, -R2a-N-, -R2a-D-, -R2a-AAN-, -R2a-AAD- or -R2a-R2b-; provided that R2a is a peptide that is cleaved at the amide bond by one or more proteolytic enzymes; R2b is a peptide that forms a carbamate with R3 by the amino group in the side chain, and the carbamate can be cleaved by one or more proteolytic enzymes; A is alanine; N is asparagine, and the amino group in its side chain forms a carbamate with R3, and the carbamate can be cleaved by Legumain; D is aspartic acid, and the amino group in its side chain forms a carbamate with R3 and can be cleaved by granzyme B. R2a and R2b can be linked by the formation of an amide bond. After Legumain and granzyme B cleave the bond (such as a carbamate) between R2 and R3, under acidic conditions, L in R3-L is cleaved, and an antibody having an R4 group is released. In some embodiments, the suitable polypeptide that can be activated by a proteolytic enzyme may be a tripeptide. In a pathological microenvironment well known in the art, any substrate peptide that is recognized and cleaved (activated) by a proteolytic enzyme can be used as R2 disclosed herein. Such peptides have the structures disclosed in WO2016 / 026458, the entire contents of which are incorporated herein by reference. In some embodiments, in the tripeptide structure suitable for the present invention, the amino acid residue linked to R1 may be selected from Ala, Thr, Val and Ile, the middle amino acid residue may be selected from Ala, Thr, Val and Asn, and the amino acid residue linked to R3 may be selected from Asn and Asp. Usually, R2 is linked to R1 by an amide bond, ester bond, urethane bond, urea bond or hydrazone bond by the amino group of its amino acid residue, and is linked to R3 by an amide bond, ester bond, urethane bond, urea bond or hydrazone bond by the carboxy group of its amino acid residue.In some preferred embodiments of the present invention, R2 is a tripeptide selected from Ala-Ala-Asn and Ala-Ala-Asp. Ala-Ala-Asn is recognized and cleaved by Legumain, and Ala-Ala-Asp is recognized and cleaved by Granzyme.

[0042] In the present invention, the R3 group is a spacer arm, including but not limited to -HN-phenyl-R’-, provided that R’ is a bond. Preferably, the NH group and R’ are located at the para-position of the phenyl group. When preparing the compound of the present invention, the R3 spacer arm of the present invention may be prepared with the compounds shown in the following formulas R3-1 and R3-2: TIFF2025520468000005.tif36170

[0043] In some embodiments, the L group is a chemical bond activated by acidity in the pathological microenvironment. Specifically, it is a hydrazone bond (i.e., -C(R’)=N-NH-, where R’ is H or a C1-4 alkyl group). For example, in some examples, the structure of R1-R2-R3-L-R4 is shown as follows: TIFF2025520468000006.tif2153.

[0044] It should be understood that the L group is obtained by the chemical reaction of the R3 compound and the R4 compound.

[0045] In the present invention, the R4 group is a binding group capable of restoring, maintaining, decreasing or promoting the binding ability between the anti-CTLA-4 antibody and its antigen, ligand or receptor after cleaving the R2 group and the R3 group. In some embodiments, after cleaving the R2 group and the R3 group, the obtained R4-s-Cys-R5 exhibits an affinity of >60% for the antigen, ligand or receptor of the natural R5.

[0046] Suitable R4 groups are represented by the following formula: TIFF2025520468000007.tif33170

[0047] However, Rc is selected from a C1-12 alkylene group, a C1-12 alkylene-O-C1-12 alkylene group, a C1-12 alkylene-C3-8 cycloalkyl group, a (C1-4 alkylene-O)p-C1-12 alkylene group, a C1-12 alkylene carbonyl amino-(C1-4 alkylene-O)p-C1-12 alkylene group, a -phenyl-C1-12 alkylene group, a C3-8 cycloalkyl group, a C1-12 alkylene-C3-8 cycloalkyl-C1-12 alkylene group, a C1-12 alkylene-NHCO-O-C1-12 alkylene group, a C1-12 alkylene-COO- and a C1-12 alkylene-phenyl-C1-12 alkylene group; p is an integer from 1 to 10, preferably 1, 2, 3 or 4; R 4-c is a bond or -CO-; However, the L group is connected to the R4 group through the R of the R4 group 4-c and is linked to the R4 group.

[0048] Generally, the R4 group binds to the S atom of cysteine of R5 via maleimide.

[0049] In a preferred embodiment, Rc is a C1-12 alkylene group and R 4-c is -CO-; however, the R4 group is linked to the L group by its carbonyl group.

[0050] In some examples, the R4 group is prepared from an R4 compound selected from the following: TIFF2025520468000008.tif103170

[0051] In the present invention, it should be understood that the wavy lines used in the various formulas shown indicate the connection positions between the parts containing the wavy lines and other parts.

[0052] As described above, R5 represents an anti-CTLA4 antibody in which one or more amino acid residues have been mutated to cysteine. In fact, R5 is a biomolecule in which the mercapto group of the introduced cysteine lacks a hydrogen atom. Due to the deletion of the hydrogen atom of this mercapto group, R5 can be regarded as a group that links to R4 of the present invention.

[0053] In one or more embodiments of the present invention, the anti-CTLA-4 antibody can be activated in multiple ways. One is to cleave R1-R2 from R3-L-R4-S-cys-R5 by a proteolytic enzyme. Another is to cleave R1-R3-L under acidic conditions in the pathological microenvironment to release R4-S-cys-R5. According to the present invention, it has been found that the dual-activation (enzyme activation and acid activation) compound of the present invention has a higher activation efficiency in diseased tissues compared to single-activation compounds.

[0054] The conjugate of the present invention can be produced by including a method of reducing the mutant biomolecule with DTT, TCEP or other reducing agents; oxidizing with Cu2SO4, dehydroascorbic acid or other oxidants; and then binding the oxidized biomolecule (R5) to R1-R2-R3-L-R4 under liquid phase or solid phase conditions. The final product can be collected in the liquid phase.

[0055] Therefore, in addition to the conjugate, the present invention further includes a functional part, namely R1-R2-R3-L-R4; R2-R3-L-R4; R3-L-R4-S-cys-R5; R4-S-cys-R5 and the mutant biomolecule; provided that R1, R2, R3, L, R4, R5 and their linking methods and the mutant biomolecule are defined in any part or any embodiment of the present invention.

[0056] In some embodiments, the functional parts are shown in CP3, CP7, CP13, CP23-CP27. In the present invention, -S-cys- indicates that the mercapto group of the cysteine introduced by R4 covalently binds to R5 through the mutation in R5. R4-S-cys-R5 is a conjugate formed under the acidic conditions of the pathological microenvironment.

[0057] The conjugates, functional moieties, R2-R3-L-R4, R3-L-R4-S-cys-R5 and R4-S-cys-R5 described in this specification can be synthesized by methods known in the art. For example, they can be prepared by the methods described in Example 1 of this application.

[0058] The present invention also includes a pharmaceutical composition containing the conjugate described in this specification. The pharmaceutical composition may further contain a pharmaceutically acceptable carrier. The carrier may be any pharmaceutically acceptable carrier or excipient, and may vary depending on the dosage form and administration method. Pharmaceutically acceptable carriers are usually safe and non-toxic, and may include known substances used in the pharmaceutical industry to prepare pharmaceutical compositions, such as fillers, diluents, aggregating agents, binders, lubricants, glidants, stabilizers, coloring agents, wetting agents, disintegrants, etc. Suitable pharmaceutically acceptable carriers include sugars such as lactose, sucrose, mannitol and sorbitol; cellulose preparations and / or calcium phosphates such as tricalcium phosphate and calcium hydrogen phosphate; starches including corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone; silica, talc, stearic acid or its salts, such as magnesium stearate or calcium stearate; and / or polyethylene glycol, etc. When selecting a pharmaceutically acceptable carrier, the administration method of the pharmaceutical dosage form is mainly considered. This is well known in the art.

[0059] The pharmaceutical composition may contain a therapeutically or prophylactically effective amount of the conjugate. "Effective amount" indicates that the amount of the component is sufficient to cause the necessary reaction. The specific effective amount depends on various factors such as the specific disease to be treated, the physical condition of the patient such as weight, age, gender, the duration of treatment, the treatment by co - administration (if any), and the specific prescription used. Usually, the "effective amount" described in this specification is a normal amount of biomolecule. However, in some embodiments, the therapeutically or prophylactically effective amount of the conjugate contained in the pharmaceutical composition of the present invention may be less than the normal biomolecule amount, but a better therapeutic or prophylactic effect can be obtained because the biomolecule is protected by a protecting group before reaching the pathological microenvironment and binding to its ligand or receptor.

[0060] The pharmaceutical composition of the present invention can be formulated into various suitable dosage forms such as tablets, capsules, injections, etc., and can be administered by any suitable method to achieve the purpose. For example, it can be administered parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, transdermally, orally, intrathecally, intracranially, intranasally or externally. The dosage of the drug depends on the age, health status and weight of the patient, the treatment carried out simultaneously, the frequency of treatment, etc. The pharmaceutical composition of the present invention can be applied to any subject having this need, for example, mammals, especially humans.

[0061] Among tumor patients, tumor cells or antigen - presenting cells (APCs) having tumor antigens bind to T cells, thereby partially or completely suppressing the immune killing of the host's tumor. However, the conjugate of the present invention is activated and released via proteolytic enzymes, especially Legumain or granzyme, and / or under acidic conditions in the pathological microenvironment.

[0062] Therefore, the conjugate of the present invention can effectively break through the individual's immune barrier, reach the pathological microenvironment, and be activated and released in the pathological microenvironment. As a result, by selectively promoting the proliferation and killing effects of T cells and the like in tumors and inflammatory microenvironments, low autoimmunity and high efficacy can be achieved.

[0063] Accordingly, various conjugates and R4-S-cys-R5 or mutated biomolecules disclosed in the present invention may be used for the treatment of tumors and inflammation, or may be used as active ingredients for modulating drugs for treating tumors and inflammation. The tumors or inflammation described in this specification are preferably tumors or inflammation mediated by CTAL-4, and tumors or inflammation known to be treatable with anti-CTAL4 antibodies (especially anti-CTLA4 antibodies described in this specification, such as Ipilimumab), which may include melanoma, bladder, brain, breast, cervical, colorectal, esophageal, kidney, liver, lung, nasopharyngeal, pancreatic, prostate, skin, stomach, uterine, ovarian, testicular cancers and cancers in the blood, etc., but are not limited thereto. Specifically, cancers include bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, uterine cancer, ovarian cancer, testicular cancer and blood cancer.

[0064] It also includes a method for treating or preventing tumors and inflammation, which includes administering a therapeutically or prophylactically effective amount of the conjugate or its pharmaceutical composition described in this specification to a subject in need. The method can be used in combination with known radiotherapy or immunotherapy.

[0065] It should be understood that the terms "comprising" and "containing" or similar expressions used in the present invention also mean "consisting of", etc. The sum of all weight % or volume % should be 100%. Unless otherwise specified, various reagents and products used in the examples are commercial products. Unless otherwise specified, the methods described in the examples are implemented based on the prior art. The following examples are not intended to limit the scope of the present invention.

[0066] Example 1: Synthesis of Functional Groups CP1-CP30 When R1, R2, R3, L and R4 are selected from different groups, the synthesized compounds are shown in Table 1-1 and Table 1-2 below: Table 1-1: Compound Names and R1, R2, R3, R4 Compounds for Forming Compounds TIFF2025520468000009.tif255169TIFF2025520468000010.tif10170

[0067] Table 1-2: Compound Structure JPEG2025520468000011.jpg217170JPEG2025520468000012.jpg242170JPEG2025520468000013.jpg234170JPEG2025520468000014.jpg178170

[0068] When R2 is Ala-Ala-Asn and R3 is PABC(R3-1), the synthetic route map is as follows: TIFF2025520468000015.tif99170

[0069] Taking CP5 as an example, the synthetic route is as follows: TIFF2025520468000016.tif137170

[0070] 1) Fmoc-Asn(Trt)-OH (20 g, 0.03 mol), 2-(7-Azabenzotriazole)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HATU) (15 g, 0.04 mol) and DMF (200 mL) were added to a three-necked flask and stirred for 30 minutes. p-Aminobenzyl alcohol (4.1 g, 0.03 mol) and N,N-diisopropylethylamine (8.7 g, 0.06 mol) were added at 0 °C respectively, and then stirred at room temperature for 3 hours. Most of the DMF was removed by rotary evaporation. The residue was dissolved in ethyl acetate (200 mL), washed successively with saturated ammonium chloride solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate, and then filtered. The solvent was removed by evaporation, and the crude product was slurried to obtain Fmoc-Asn(Trt)-PABC as a white solid (21.3 g; yield: 90%).

[0071] 2) Fmoc-Asn(Trt)-PABC (16.0 g, 22 mmol) was dissolved in N,N-dimethylformamide (80 mL). Piperidine (30 mL) was added, and the mixture was stirred at room temperature for 2 hours. The solvent was removed by evaporation under reduced pressure. The residue was dried in a vacuum oven under high vacuum to remove a small amount of piperidine, and 9.8 g of NH2-Ann(Trt)-PABC, a pale yellow solid, was obtained and could be used in the next step without purification.

[0072] 3) Alloc-Ala-Ala-OH (5.0 g, 20.4 mmol), benzotriazol-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HBTU) (11.6 g, 30.6 mmol), and DMF (50 mL) were added to a three-necked flask and stirred in an ice bath for 30 minutes. At 0 °C, NH2-Asn(Trt)-PABC (9.8 g, 20.4 mmol) and N,N-diisopropylethylamine (7.89 g, 61.2 mmol) were added respectively, and then the mixture was stirred at room temperature overnight. The solvent was removed by evaporation under reduced pressure. The residue was dissolved in ethyl acetate (200 mL), washed successively with saturated ammonium chloride solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by evaporation. The obtained crude product was recrystallized to obtain Alloc-AAN(Trt)-PABC (13.0 g; yield: 90%) as a white solid.

[0073] 4) Alloc-AAN(Trt)-PABC (10.0 g, 14.2 mmol) was dissolved in dichloromethane (100 ± 15 mL). Trifluoroacetic acid (20 mL) was added, and the mixture was stirred at room temperature for 4 hours. After washing with water and fractional distillation, the organic phase was dried over anhydrous sodium sulfate. The solvent was removed by evaporation under reduced pressure, and the remaining trifluoroacetic acid was removed by high vacuum evaporation. The crude product was separated by column chromatography to obtain Alloc-AAN-PABC (5.9 g; yield: 89%).

[0074] 5) Alloc-AAN-PABC (467 mg, 1.01 mmol) dissolved in dichloromethane (10 mL) was added to a three-necked flask. Under an ice bath and protected by nitrogen gas, 4-nitrophenyl chloroformate (406 mg, 2.02 mmol) in dichloromethane and pyridine (160 mg, 2.03 mmol) in dichloromethane were each added dropwise to the flask, and the mixture was stirred at room temperature overnight. 1-(6-Aminohexyl)-1H-pyrrole-2,5-dione (235 mg, 1.2 mmol) was added to the above solution in one batch, and the reaction was carried out at room temperature for 4 hours. The reaction solution was dried by rotary evaporation. The obtained crude product was purified by silica gel column chromatography to obtain S15-1 (540 mg; yield: 80%) as a white solid.

[0075] 6) DMF (10 mL), S15-1 (208 mg, 0.31 mmol), acetic acid (274 mg, 4.65 mmol), triphenylphosphine (72 mg, 0.062 mmol) and tributyltin hydride (1.17 g, 4.03 mmol) were sequentially added to a single-necked flask. After replacing the air in the flask with nitrogen gas, the mixture was stirred at room temperature until S15-1 was completely reacted. After the reaction was completed, DMF was removed by evaporation under reduced pressure. The crude product was separated and purified by silica gel column chromatography to obtain S15-2 (white solid, 116 mg, yield: 62%).

[0076] 7) Compound 1 (940 mg, 0.18 mmol), benzotriazol-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) (95 mg, 0.25 mmol) and DMF (10 mL) were added to a three-necked flask, and then stirred in an ice bath for 30 minutes. Then, at 0 °C, compound intermediate 2 (108 mg, 0.18 mmol) and N,N-diisopropylethylamine (70 mg, 0.54 mmol) were added thereto respectively, and the mixture was stirred at room temperature overnight. The solvent was removed by evaporation under reduced pressure. The crude product was separated and purified by silica gel column chromatography to obtain a white solid (85.5 mg; yield: 40%), which is compound CP5.

[0077] When R2 has the amino acid sequence of Ala - Ala - Asp and R3 is PABC, the synthetic route is as follows: TIFF2025520468000017.tif97170

[0078] CP1, CP2, CP4, CP6, CP12, CP13, CP15, CP20 - CP22, CP28 - CP30 were synthesized by the above method.

[0079] When it contains a chemical bond activatable by an acid, the synthetic scheme is as follows: TIFF2025520468000018.tif15170

[0080] As shown in CP8, the specific synthesis process is as follows: TIFF2025520468000019.tif48170

[0081] 1) R1 - 10 (3.1 g, 0.01 mol), 2-(7 - azabenzotriazole)-N,N,N’,N’ - tetramethyluronium hexafluorophosphate (HATU) (4.56 g, 0.012 mol) and DMF (20 mL) were added to a three - necked flask and stirred for 30 minutes. Respectively, at 0 °C, R3 - 2 (1.35 g, 0.01 mol) and N,N - diisopropylethylamine (3.87 g, 0.03 mol) were added, and then stirred at room temperature for 3 hours. DMF was removed by rotary evaporation. The residue was purified by silica gel column chromatography to obtain intermediate 1 (2.5 g; yield 59%) as a pale yellow oil.

[0082] 2) Intermediate 1 (98 mg, 0.23 mmol) and R4-18 (42 mg, 0.23 mmol) were sequentially weighed and added to a 50 ml single-necked flask. Dichloromethane (5 mL) was added to dissolve Intermediate 1 and R4-18, and then 4A molecular sieves (81 mg) were added. After replacing the air in the flask with nitrogen gas, the mixture was reacted overnight at room temperature. The reaction solution was dried by rotary evaporation. The crude product was separated and purified by silica gel column chromatography to obtain a white solid (81 mg; yield: 60%), which is Compound CP8.

[0083] CP7, CP9-11, CP14, and CP19 were synthesized by the above method.

[0084] Taking CP23 as an example, when the amino acid sequence of R2 is Ala-Ala-Asn and L is a chemical bond that can be activated by an acid, the synthesis scheme is as follows: TIFF2025520468000020.tif160170

[0085] 1) Synthesis of Intermediate 1 Take a dry and clean 2 L reaction flask, add 500 ml of THF, weigh 80 g of Fmoc-Asn(Trt)-OH, add it to the reaction flask, stir to dissolve, add 46.6 g of DEPBT, stir at room temperature for 15 minutes, add 16 g of PABC, react at room temperature for 30 minutes, then add 45 ml of DIPEA, replace the air with nitrogen gas for protection, react at room temperature for 3 hours, and monitor the end of the reaction (complete reaction of Fmoc-Asn(Trt)-OH) by TLC. The reaction solution was distilled off under reduced pressure, a small amount of DMF (180 mL) was added to dissolve it, and it was dropped into 3 L of stirring water to precipitate a pale yellow solid. After washing with water 2-3 times, it was suction filtered, the solid was collected, and vacuum dried to obtain an off-white solid (yield over 90%).

[0086] 2) Synthesis of Intermediate 2 500 mL of THF and the off-white solid obtained in the previous step were successively added to a 2 L single-neck reaction flask, stirred and dissolved, cooled to 0 - 5 °C in an ice-salt bath, 100 mL of piperidine was added dropwise. After the addition was completed, the temperature was gradually returned to room temperature and reacted for 1 hour. The reaction was monitored by TLC until completion. The solvent was distilled off under reduced pressure, a small amount of DMF was added and dissolved, and the solution was added dropwise to 2 L of stirred water, mechanically stirred for 30 min, suction filtered, washed with water 2 - 3 times repeatedly, suction filtered again. 800 mL of methyl tert-butyl ether was added to the cake, stirred for 30 min, suction filtered, PE:EA = 10:1 was added to the cake for washing twice, suction filtered, the cake was collected, and after vacuum drying, 80 g of an off-white solid was obtained with a purity of 70%.

[0087] 3) Synthesis of Intermediate 3 To a dry and clean 250 mL single-neck reaction flask, 50 mL of THF, 5.04 g of Boc-Ala-Ala-OH, and 3.89 g of DEPBT were successively added, reacted at room temperature for 10 min, 2.6 g of NH2-Asn(Trt)-PABC was added, the air was exchanged and protected with nitrogen gas, reacted at room temperature for 15 min, 3.5 mL of DIPEA was added dropwise, the air was exchanged and protected with nitrogen gas, reacted at room temperature for 3 hours, the solvent was distilled off under reduced pressure, water was added to make a slurry 2 - 3 times, suction filtered, and a light yellow solid was obtained, 3.7 g. It was purified by passing through a column to obtain 2.0 g of the product with a purity of 94.8% and a yield of 26.6%.

[0088] 4) Synthesis of Intermediate 4 1.8 g of BOC-Ala-Ala-ASN(TRT)-PABC was weighed and added to a 100 mL single-neck reaction flask, 28.5 mL of TFA was added, 1.5 mL of water was added dropwise, reacted at room temperature for 30 min, the reaction was monitored by TLC until completion, the solvent was distilled off under reduced pressure, methyl tert-butyl ether was added to make a slurry, suction filtered to obtain a solid. A solution of dioxane:water = 1:1 was added to dissolve it, 1 N sodium hydroxide was added to adjust the pH to 13, stirred at room temperature for 40 min, the solvent was distilled off under reduced pressure, mixed with silica gel, passed through a column, and 450 mg of the product was obtained. The yield was 47.5%.

[0089] 5) Synthesis of Intermediate 5 In a 500 ml three-necked reaction flask, add 100 ml of dioxane, 9.8 g of NH2-AAN-PABC, 3.28 g of Na2CO3 (added dropwise as a saturated aqueous solution), cool the temperature to 0 - 5 °C in an ice-salt bath, weigh 6.6 g of Fmoc-Cl, add 10 ml of dioxane to dissolve it, add it dropwise to the reaction solution, gradually warm up to room temperature and react for 30 min (precipitate solids, add THF to continue the reaction), until the reaction is completed, distill off the solvent under reduced pressure, add water to make a slurry, filter by suction to obtain an off-white solid, add methyl tert-butyl ether to make a slurry, filter by suction to obtain an off-white solid, vacuum dry until the weight is constant to obtain 15.5 g of the product, with a purity of 80% and a yield of 80%.

[0090] 6) Synthesis of Intermediate 6 Dissolve Intermediate 5 (5 g, 8.3 mol) in DMF (40 mL), add Dess-Martin reagent (5.3 g, 12.5 mmol) while stirring at room temperature, and continue stirring for 2 hours. Quench the reaction solution with saturated Na2S2O4 solution and saturated NaHCO3 solution, filter, concentrate the filtrate under reduced pressure, wash the solid with water to obtain Intermediate 6 (4.7 g of a brown solid, yield: 94%).

[0091] 7) Synthesis of Intermediate 7 Dissolve Intermediate 6 (566 mg, 0.94 mmol) in DMF (10 mL), add Compound 2 (280 mg, 0.94 mmol) while stirring, and stir overnight at room temperature under the protection of nitrogen gas. Prepare the reaction solution under medium pressure to obtain Intermediate 7 (719 mg of a white solid, yield: 100%).

[0092] 8) Synthesis of Intermediate 8 Dissolve Intermediate 7 (280 mg, 0.37 mmol) in DMF (10 mL), add DBU (5 μl) while stirring, stir at room temperature for 3 hours, then add DBU (5 μl) and continue stirring for 2 hours. Prepare the reaction solution under high pressure to obtain Intermediate 8 (85 mg of a white solid, yield: 42%).

[0093] 9) Synthesis of the final product CP23 Compound SCM-5K (1.45 g, 0.29 mmol) was dissolved in DMF (20 ml), and Intermediate 8 (190 mg, 0.35 mmol) was added while stirring, followed by stirring overnight at room temperature. The reaction solution was concentrated under reduced pressure and purified by high-performance liquid chromatography to obtain Product CP23 (1.12 g of white solid, yield: 77%).

[0094] CP3, CP5, CP16 - CP18, CP24 - CP27 were synthesized by the above method.

[0095] Compounds CP1 - CP30 were verified by mass spectrometry (MS), and their molecular weights were shown in Table 2, which were consistent with the molecular weights calculated based on their structures.

[0096] Table 2: Mass spectrometry information table of the functional substrates of the present invention and their properties and yields TIFF2025520468000021.tif255170TIFF2025520468000022.tif116170

[0097] Example 2: Construction of a series of anti - hCTLA4 TMEAbody with different blocking groups and linkers A series of anti - human CTLA4 - TMEAbody (TMEAbody is an abbreviation for antibodies activated by the tumor microenvironment) were constructed by site - specific coupling of different compounds (including inhibitory groups and linkers) with Ipilimumab containing the Y49C (Y49C is in a different Kabat numbering system for antibodies, and this number corresponds to Y50C which should be numbered sequentially) mutation in the light chain. The Ipilimumab antibody containing Y49C (abbreviated as Ipi - L - 3 here) was reduced with dithiothreitol (DTT) at a molar ratio of 1:200 for 16 h under the conditions of 50 mM Tris (pH 7.5), 2 mM EDTA at room temperature, then dialyzed in 50 mM Tris (pH 7.5), 150 mM NaCl buffer, and then re - oxidized with dehydroascorbic acid (DHAA) at a molar ratio of 1:100 and used to couple with different compounds CP12 - CP18, CP23 - CP30 containing different inhibitory groups and different chemical linkers. The molar ratio of the compound / antibody used for coupling is 1:20.

[0098] To remove excess compounds, the coupled TMEAbody was further purified by a Protein A column, and the coupling efficiency was evaluated by SDS-PAGE measurement. From the results in Figure 1 (A-F), it was found that Ipi-L-3 showed high coupling efficiency for these different compounds.

[0099] Example 3: ELISA Characterization of Ipi-L-3 TMEAbody To characterize the binding properties of the constructed TMEAbody and human CTLA-4 protein, 0.5 μg / ml of His-tagged human CTLA-4 protein (Sino Biological, Cat#11159-H08H) was coated on a Maxisorp ELISA plate (Nunc) and incubated overnight at 4°C. Then, the plate was washed three times with PBST and blocked with 2% BSA for 2 hours at room temperature. After washing the ELISA plate three times with PBST, it was incubated for 1 hour with a series of concentrations of the TMEAbody after coupling, the (cysteine mutant) antibody before TMEAbody coupling, and the control wild-type Ipilimumab antibody. Then, the plate was washed three times with PBST, and an anti-human Fc antibody conjugated with HRP (Invitrogen, Cat#A18829) was incubated at a dilution of 1:5000 for 0.5 hour at room temperature. After washing three times with PBST, the plate was developed with tetramethylbenzidine (TMB, solarbio, catalog number PR1200) and ELISA stop buffer (solarbio, catalog number C1058). Then, the absorbance at 450 nm was measured with an ELISA microplate reader (biotek, ELx800 Absorbance Microplate Reader). Then, the data was analyzed with GraphPad Prism 5 software. The results of the ELISA are shown in Figure 2 (A-F). These results showed that the binding ability of the TMEA antibody and CTLA-4 protein was blocked by different blocking groups at different levels. Usually, the blocking efficiency increases with the size of the blocking group.

[0100] Example 4: Activation of Ipi-L-3+CP23, Ipi-L-3+CP24, Ipi-L-3+CP25 and Ipi-L-3+CP26 TMEAbody Mediated by Acid Compounds CP23, CP24, CP25 and CP26 have hydrazone bonds that are unstable at acidic pH. The Ipi-L-3 TMEAbody coupled with these compounds was dialyzed against citrate buffer at pH 4.5, pH 5.0, and pH 6.0, and acid-mediated cleavage was monitored by SDS-PAGE. As shown in Figures 3(A - B), all of the TMEAbodies with these compounds showed varying degrees of cleavage, and the degree of cleavage depended on the pH value, with higher cleavage efficiency at lower pH.

[0101] Subsequently, ELISA measurements were performed by the method of Example 2 to evaluate the recovery of binding activity. The results are summarized in Figures 4(A - D). Acid-mediated cleavage at low pH caused corresponding recovery of activity, and the level of activity recovery was proportional to the cleavage level.

[0102] Example 5: Activation of Ipi-L-3+CP23 TMEAbody Mediated by Legumain The coupled Ipi-L-3+CP23 TMEAbody and activated Legumain were incubated overnight at 37 °C in a buffer of pH 6.0, 50 mM MES, and 250 mM NaCl. Protein blotting was used to evaluate the activation efficiency. The light chain of the antibody was detected using an anti-human Fab HRP antibody (SIGMA, A0293). As shown in Figure 5, 1 μg of IPI-L-3+CP23 tmeabody can be activated by 1 - 5 μg of Legumain.

[0103] Subsequently, the above ELISA test was performed to measure the recovery of binding ability after Legumain digestion. The results are shown in Figure 6. As a result, it was shown that different degrees of Legumain cleavage led to corresponding recovery of activity.

[0104] Example 6: ELISA Characterization of Acid-Activated and Enzyme-Activated Figure 7 shows the image of the activation of Legumain of the TMEAbody in which Ipi-L-3 is coupled with CP23 by acid. To investigate the activity recovery after activation by acid, R4-4 was used to couple with Ipi-L-3, and the molecular structure is the same as that after acid-mediated activation.

[0105] After coupling with R4-4, the influence of the small molecule fragment remaining in the activated antibody on the Ipi-L-3 binding activity was evaluated by ELISA. The results are shown in Figure 8(A), indicating that after Ipi-L-3 is coupled with R4-4, the binding activity of Ipi-L-3 is maintained, meaning that the structure after activating Ipi-L-3+CP23 by acid can maintain the same binding activity as the wild-type Ipilimumab antibody.

[0106] To investigate the activity recovery after activation by enzyme, CP31 was used to couple with Ipi-L-3, and the molecular structure is the same as that after enzyme-mediated activation. After coupling with CP31, the influence of the small molecule fragment remaining in the activated antibody on the Ipi-L-3 binding activity was evaluated by ELISA. The results are shown in Figure 8(B), indicating that after Ipi-L-3 is coupled with CP31, the binding activity of Ipi-L-3 is maintained, meaning that the structure after activating Ipi-L-3+CP23 by Legumain can maintain the same binding activity as the wild-type Ipilimumab antibody.

[0107] Example 7: Evaluation of the Rapid Acid-Activation and Activity Recovery of Ipi-L-3+CP23 To establish a rapid acid-activation method for Ipi-L-3 CP23 TMEAbody, after coupling, the pH values of Ipi-L-3 CP23 TMEAbody were adjusted to pH 2.0, pH 2.5, and pH 3.5 with HCl, and treated for 1 h, 3 h, and 5 h, respectively. At the same time, SDS-PAGE and ELISA were used to evaluate the cleavage efficiency and activity recovery.

[0108] The results are shown in Table 9 and Figures 10(A-D). As a result, it was found that the TMEAbody could be rapidly cleaved under extremely low pH conditions such as pH 2.0 and pH 2.5. However, the ELISA results showed that extremely low pH led to a significant loss of antibody activity, which might be due to the disruption of the antibody structure by extremely low pH. Also, the wild-type Ipilimumab antibody showed a significant loss of activity even under the conditions of pH 3.5, pH 2.5, and pH 2.0.

[0109] Example 8: Evaluation of the Pharmacokinetics and Tissue Distribution of a Series of TMEAbody in a Mouse Tumor Model To evaluate the distribution and activation of TMEAbody in vivo, the above series of 10 mg / kg TMEAbody was injected into CT26-bearing Balb / c mice, and then plasma and different tissues were collected at different time points and used for the analysis of the concentration of TMEAbody or activated TMEAbody. Tissues were homogenized in 2.5 volumes of PBS and 1X Halt TMProtease Inhibitor Cocktail (100x) (Thermo Fisher, catalog number 78430) was added, and the supernatant was collected and used for ELISA testing. For measuring the TMEAbody concentration, the TMEAbody was captured with a 5 μg / ml anti-PEG antibody (Abcam, catalog number ab51257), and the captured TMEAbody was detected with an anti-human Fc antibody (Invitrogen, A18829, diluted 1:50000). A calibration curve was created using a series of TMEAbody with different concentrations. The homogenized supernatants of plasma and tissue were diluted by an appropriate multiple and used for ELISA measurement, and the concentration was converted using the calibration curve. To measure the concentration of active TMEAbody, it was coated with 0.5 μg / ml human CTLA-4 protein (Sino Biological, 11159-H08H), and a series of concentrations of wild-type Ipilimumab was used as the primary antibody, and a 1:5000 anti-human Fc antibody (Invitrogen, catalog number A18829) was used as the secondary antibody to create a calibration curve using different concentrations of the Ipilimumab antibody. The homogenized supernatants of plasma and tissue were diluted by an appropriate multiple and used for ELISA measurement, and the concentration was converted using the calibration curve.

[0110] The inventors focused on two important parameters in the PK and tissue distribution data of TMEAbody. One parameter is the activation rate in tumors and other tissues. Due to low peripheral toxicity, a high specific activation rate in tumors rather than in other tissues is preferred. The activation rates of TMEAbody in different tissues are shown in Table 3.

[0111] Table 3: Activation efficiency of TMEAbody at different times (days) in different tissues TIFF2025520468000023.tif255169TIFF2025520468000024.tif167170

[0112] Since day D0 is the starting point of administration, ND indicates that there is no data in the organ on that day and only data in the blood.

[0113] As shown in the results of Table 3, Ipi-L-3+CP23 / CP24 / CP25 / CP26 / CP28 / CP30 shows different degrees of priority of tumor activation, and the activation efficiency decreases with the increase of the blocking group. The linkers (CP23, CP24, CP25, CP26) activated by legumain and acid show higher activation efficiency than the linkers activated only by legumain (CP28, CP30).

[0114] Another parameter that the inventors focus on is tumor permeability, because PEG can increase the size of the antibody and may affect the tumor permeability of the antibody. The permeability was expressed as the tumor AUC / blood AUC (total concentration of activated and inactivated TMEAbody, TMEABODY) ratio. As shown in Figure 11, PEG with a molecular weight greater than 20 kD significantly affects the permeability of the antibody.

[0115] Example 9: Therapeutic effect of Ipi-L-3 TMEAbody in an MC38 mouse tumor model with hCTLA-4 gene knocked in To evaluate the in vivo therapeutic effect of Ipi-L-3 TMEAbody on mouse tumors, Ipi-L-3+CP23 TMEAbody, wild-type Ipilimumab, and control human IgG were administered to a C57BL / 6 mouse (Shanghai Model Organisms) MC38 colon adenocarcinoma tumor model with the human CTLA-4 gene knocked in. 2E6 MC38 cells were subcutaneously injected into the left lower abdominal quadrant of C57BL / 6 mice with the human CTLA-4 gene knocked in. Seven days after the tumors grew, the animals were grouped according to the same average tumor volume. Twice a week, the animals were administered the designated dose of control human IgG, wild-type Ipilimumab, or IPI-L-3+CP23 TMEAbody (antibody concentration without PEG molecular weight, n = 6), and the tumor volume of each animal was monitored.

[0116] As a result, as shown in FIGS. 12(A-E), the Ipi-L-3+CP23 TMEAbody shows a dose-dependent tumor growth inhibitory effect. When the dose is 0.5 mg / kg, the Ipi-L-3+CP23 TMEAbody shows the same complete remission rate (CR) as the WT Ipilimumab antibody.

[0117] Example 10: The Ipi-L-3 TMEAbody showed an extended serum half-life in mice knocked-in with the human CTLA-4 gene Antibodies show target antigen-mediated clearance in the body. Since the binding affinity of TMEAbody to the target antigen decreases, the target antigen-mediated clearance effect is inhibited, and it is presumed that the half-life becomes longer. Wild-type Ipilimumab antibody or Ipi-L-3+CP23 TMEAbody was administered by injection to mice knocked-in with hCTLA-4 (Shanghai Model Organisms Center, Inc.), plasma was collected at different time points, and the antibody concentration was measured by "sandwich" ELISA using anti-human Fc and anti-human Fab antibodies. As shown in FIG. 13, the Ipi-L-3+CP23 TMEAbody showed an extended serum half-life and AUC compared to the wild-type Ipilimumab antibody.

[0118] Example 11: The Ipi-L-3 TMEAbody shows a reduced peripheral T cell activation effect To evaluate the activation effects of wild-type Ipilimumab antibody and Ipi-L-3+CP23 TMEAbody on peripheral CD4+ T cells, wild-type Ipilimumab, Ipi-L-3+CP23 TMEAbody or PBS was injected into mice (Shanghai Model Organisms Center, Inc.) knocked-in with hCTLA-4 at doses of 1 mg / kg, 3 mg / kg, and 10 mg / kg, respectively. At the designated time points, blood was collected and Ki67 of CD4+ T cells was monitored as a proliferation marker. As shown in Figure 14 (A-C), Ipi-L-3+CP23 TMEAbody did not stimulate the proliferation of CD4+ T cells at doses of 3 mg / kg and 10 mg / kg. Since the activity of TMEAbody was blocked, it was suggested that TMEAbody had a low effect on activating peripheral blood T cells and had lower immune-related toxicity.

[0119] Example 12: Ipi-L-3 TMEAbody caused a low GvHD effect in NSG mice transplanted with human PBMC To further evaluate the immunotoxicity of Ipilimumab and Ipi-L-3 TMEAbody, wild-type Ipilimumab and Ipi-L-3+CP23 TMEAbody were administered to NSG immunodeficient mice (Shanghai Model Organisms Center, Inc.) transplanted with human PBMC. 5×10 6 human PBMC cells were transplanted into NSG mice via the tail vein and reconstructed for 10 days, and then administered with 10 mg / kg Ipilimumab or Ipi-L-3 TMEAbody twice a week. As an indicator of graft-versus-host disease (GvHD effect), the body weight of the animals was observed. As can be seen from Figure 15, wild-type Ipilimumab clearly exacerbated GvHD and caused a rapid decrease in body weight, while Ipi-L-3+CP23 TMEAbody did not clearly exacerbate GvHD, suggesting that Ipi-L-3+CP23 reduced the activation of toxic T cells in healthy tissues.

[0120] Example 13: Ipi-L-3 TMEAbody caused low immunogenicity in mice To evaluate the immunogenicity of Ipi-L-3 TMEAbody, Ipi-L-3+CP23 TMEAbody and WT CTLA-4 antibody were injected into Balb / c mice for immunization, with PBS as the negative control. For the primary immunization, 50 μg of Ipi-L-3+CP23 TMEAbody or wild-type Ipilimumab antibody was mixed with 1:1 complete Freund's adjuvant (CFA) and injected intraperitoneally into mice, with 5 mice in each group. To boost the immunity further, on the 14th day after the primary immunization, 25 μg of Ipi-L-3 TMEAbody or wild-type Ipilimumab antibody was mixed with 1:1 incomplete Freund's adjuvant and injected intraperitoneally into mice. On the 21st day after the primary immunization, plasma was collected and immunogenicity was analyzed by ELISA. 0.5 μg / mL of human CTLA-4-His protein (Sino Biological, Cat#11159-H08H) was coated overnight on an ELISA plate (BeaverBio, Cat#REF40301). After blocking with 1% BSA, a series of concentrations of serum and 0.1078 μg / ml of wild-type Ipilimumab antibody were mixed, and 100 μl of the mixture was added to the wells of a 96-well ELISA plate on which human CTLA-4-his protein was immobilized. Goat anti-human IgG (Fab specificity) antibody conjugated to peroxidase (Sigma, Cat#A0293) was used as the secondary antibody at 1:5000, and the plate was developed with tetramethylbenzidine (TMB, solarbio, catalog number PR1200) and ELISA stop buffer. The absorbance at 450 nm was measured using an ELISA tablet reader (Biotek, ELx800 absorbance enzyme marker). The data were analyzed with GraphPad Prism 5 software.

[0121] The ELISA format is shown in Figure 16(A), and the results are shown in Figure 16(B). These results indicate that the Ipi-L-3 TMEAbody has a lower risk of inducing the production of neutralizing anti-drug antibodies (nADA) compared to the wild-type Ipilimumab antibody.

[0122] Example 14: Evaluation of the therapeutic effect of Ipi-L-3 TMEAbody on a mouse MC38 tumor model with human CTLA-4 gene knocked in by FACS To evaluate the depletion effect of mouse tumors and peripheral Treg cells after treatment with Ipi-L-3 TMEAbody, Ipi-L-3 + CP23 TMEAbody and Ipilimumab antibody were administered to C57 mice with human CTLA-4 gene knocked in and having MC38 tumors. Seven days after administration, the spleen and tumors were collected and used for FACS analysis (Beckman Coulter, CytoFLEX) of the Treg subgroup (CD4+CD25+FoxP3+). Subsequently, the data were analyzed with GraphPad Prism 5 software.

[0123] The results are shown in Fig. 17 (A - B). From these results, it was suggested that Ipi-L-3 TMEAbody decreased the percentage of Treg cells in tumors rather than in the spleen, that it was activated locally in the tumor and exerted an anti-tumor effect, and that the activation in peripheral tissues was low.

Claims

1. The following structure: R1 - R2 - R3 - L - R4 - S - cys - R5 A conjugate of an anti - CTLA - 4 antibody having the same. However, R5 represents an anti - CTLA - 4 antibody in which one or more of its amino acid residues are mutated to cysteine; cys represents the cysteine residue; S represents the S atom in the cysteine residue; R1 is a functional group that blocks the binding of R5 to its antigen, ligand or receptor; R2 is a spacer arm that can be hydrolytically cleaved by one or more proteases in the pathological microenvironment; R3 is a spacer arm that provides space for protease cleavage; L is a chemical bond activated under acidic conditions in the pathological microenvironment; preferably, L is a hydrazone bond; R4 is a group that covalently binds to R5 via the sulfur atom of the cysteine residue, and after the cleavage of R1 - R2 - R3 - L -, the binding ability of R5 to its antigen, ligand and receptor can be restored, maintained or improved.

2. R1 is NR ａ R ｂ -R-CO-, C １－４ alkoxy-(C １－４ alkoxy) ｎ -carbonyl-C １－４ alkylene-carbonyl-, The conjugate according to claim 1, characterized in that it is selected from: However: Each R is independently a C1 - 4 alkyl group; Each n is independently an integer in the range of 1 to 30000; peg ｍ is polyethylene glycol with a molecular weight of 44 to 132,000, where m represents the molecular weight of polyethylene glycol; R ａ is C １－４ alkyl - O - [(CH ２ ) ｑ - O] ｎ - (CH ２ ) ｑ - ;​ R ｂ is C １－４ alkyl-O-[(CH ２ ｑ -O] ｎ -(CH ２ ｑ -C(O)-;​​ q is an integer from 1 - 4; The wavy line indicates the position where R1 is linked to R2; Preferably, the NR ａ R ｂ -R-CO- is Said C １－４ alkoxy-(C １－４ alkoxy) ｎ -carbonyl-C １－４ alkylene-carbonyl- is Preferably, R1 is selected from the following: More preferably, R1 is the aforementioned R1 - 7, R1 - 15, R1 - 16, R1 - 17, R1 - 20 or R1 - 21.

3. R2 is a tripeptide, provided that the amino acid residue linked to R1 in the tripeptide is selected from Ala, Thr, Val and Ile, the middle amino acid residue is selected from Ala, Thr, Val and Asn, and the amino acid residue linked to R3 is selected from Asn and Asp; preferably, R2 is Ala - Ala - Asn or Ala - Ala - Asp; However, R2 is linked to R1 by an amide bond, ester bond, urethane bond, urea bond or hydrazone bond linkage method through the amino group of its amino acid residue, and is linked to R3 by an amide bond, ester bond, urethane bond, urea bond or hydrazone bond linkage method through the carboxy group of its amino acid residue The conjugate according to claim 1, characterized in that.

4. R3 is -HN-phenyl-R'- where R' is a bond; preferably, the NH group and R' are located at the para-position of the phenyl group, the conjugate according to claim 1.

5. The structure of the R1-R2-R3-L-R4 is where R' is H or a C1-4 alkyl group the conjugate according to claim 1, characterized in that.

6. R4 is where Rc is selected from a C1-12 alkylene group, a C1-12 alkylene-O-C1-12 alkylene group, a C1-12 alkylene-C3-8 cycloalkyl group, a (C1-4 alkylene-O)p-C1-12 alkylene group, a C1-12 alkylene carbonylamino-(C1-4 alkylene-O)p-C1-12 alkylene group, a -phenyl-C1-12 alkylene group, a C3-8 cycloalkyl group, a C1-12 alkylene-C3-8 cycloalkyl-C1-12 alkylene group, a C1-12 alkylene-NHCO-O-C1-12 alkylene group, a C1-12 alkylene-COO- and a C1-12 alkylene-phenyl-C1-12 alkylene group; p is an integer from 1 to 10, preferably 1, 2, 3 or 4; R ４－ｃ is a bond or -CO-; However, the L group is connected to the R4 group via the R of the R4 group; the R4 group is bonded to the S atom of the cysteine of R5 via maleimide; ４－ｃ ​ Preferably, Rc is a C1-12 alkylene group, and R ４－ｃ is -CO- the conjugate according to claim 1, characterized in that.

7. The anti-CTLA-4 antibody is ipilimumab, the antibody according to claim 1, characterized in that; Preferably, the mutation positions on the ipilimumab heavy chain are selected from Gln3, Arg19, Leu20, Ser25, Gly26, Phe27, Thr28, Phe29, Ser30, Ser31, Tyr32, Thr33, Met34, His35, Gly44, Phe50, Ile51, Ser52, Tyr53, Asp54, Gly55, Asn56, Asn57, Lys58, Tyr59, Tyr60, Ala61, Asp62, Ser63, Lys65, Gly66, Thr69, Ser71, Arg72, Asp73, Asn74, Ser75, Lys76, Asn77, Thr99, Gly100, Trp101, Leu102, Gly103, Pro104, Leu105, Asp106 and Tyr107; preferably, they are selected from Ser30, Ser31, Tyr32, Thr33, Ile51, Asp54, Gly55, Asn56, Lys58, Tyr59, Tyr60, Ala61, Asp62, Ser63, Lys65, Gly66, Gly100, Trp101, Leu102, Gly103, Pro104, Leu105, Asp106 and Tyr107; Preferably, the mutation positions in the ipilimumab light chain are selected from Gln6, Arg24, Ala25, Ser26, Gln27, Ser28, Val29, Gly30, Ser31, Ser32, Tyr33, Leu34, Ala35, Tyr37, Ile49, Tyr50, Gly51, Ala52, Phe53, Ser54, Arg55, Ala56, Thr57, Gly58, Ile59, Pro60, Asp61, Arg62, Ser68, Gly69, Thr70, Gln90, Gln91, Tyr92, Gly93, Ser94, Ser95, Pro96, Trp97, Thr98, Phe99 and Gly100; preferably, they are selected from Ala25, Ser26, Ser28, Gly30, Ser31, Ser32, Leu34, Ala35, Gly51, Ala52, Ser54, Ala56, Thr57, Gly58, Ile59, Gly93, Ser94, Ser95, Thr98 and Gly100; The position numbers refer to SEQ ID NOs: 1 and 2; Preferably, Tyr50 of the ipilimumab light chain mutates to C. [

8. ] The R1-R2-R3-L-R4 is The conjugate according to claim 1, characterized in that it is selected from

9. A drug composition comprising the conjugate according to any one of claims 1-8 and a pharmaceutically acceptable carrier.

10. Use of the conjugate according to any one of claims 1-8 in the preparation of a drug for treating or preventing a tumor or inflammation; preferably, the tumor or inflammation is a tumor or inflammation that can be treated or prevented with an anti-CTLA-4 antibody; more preferably, the tumor is selected from bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, uterine cancer, ovarian cancer, testicular cancer and blood cancer.

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