Patent Description:
B7-H3, also known as CD276, was first reported in <NUM> (<NPL>), and it is considered not to belong to butyrophilin and myelin oligodendrocyte glycoprotein because the protein thereof lacks a heptad structure and B30. <NUM> domain, and it is identified as a member of the immunoglobulin superfamily, B7 family (<NPL>), and it differs from other members of the family such as PD-L1, B7-H4, CD80, CD86 in that B7-H3 exists in the human body in the form of two different variants, namely 2IgB7-H3 and 4IgB7-H3, wherein 4IgB7-H3 is an exon duplication of 2IgB7-H3, which mainly exists in the form of 4IgB7-H3 in human body (<NPL>; <NPL>; <NPL>), while only 2IgB7-H3 structure is contained in mice (<NPL>). The results of the study showed that the 2IgB7-H3 of the natural mouse and the 4IgB7-H3 of the human show similar functions without functional differences (<NPL>; <NPL>. ), the crystal structure shows that the FG loop of the IgV region of the protein is an important epitope for serving the function of B7-H3 (<NPL>).

Although the mRNA level of B7-H3 is widely expressed, for example, high B7-H3 mRNA expression level can be detected in many tissues and organs of the human body, including heart, liver, placenta, prostate, testis, uterus, pancreas, small intestine, and colon. However, the protein expression level is relatively limited to non-immune cells such as resting fibroblasts, endothelial cells, osteoblasts, amniotic fluid stem cells, as well as the surface of induced antigen-presenting cells and NK cells (<NPL>; <NPL>; <NPL>). The protein level of B7-H3 is lowly expressed in normal healthy tissues. For example, the low protein level of B7-H3 can be detected in tissues such as liver, lung, bladder, testis, prostate, breast, placenta, and lymphoid organs of normal humans, but B7-H3 protein is overexpressed in a large number of malignant tumors and is a marker antigen of tumor cells. Studies have shown that B7-H3 can be highly expressed in many cancers such as prostate cancer, ovarian cancer, colorectal cancer, renal cell carcinoma, non-small cell lung cancer, pancreatic cancer, melanoma, gastric cancer, bladder cancer, malignant glioma, and osteosarcoma, particularly highly and abnormally expressed in many cancers such as head and neck cancer, renal cancer, brain glioma, and thyroid cancer (<NPL>; <NPL>; <NPL>; <NPL>; <NPL>; <NPL>; <NPL>; <NPL>; <NPL>; <NPL>; <NPL>). B7-H3 is not only expressed on tumor cells, but it is also highly expressed on tumor neovascular endothelial cells, and is a very broad-spectrum tumor marker antigen. High expression of B7-H3 protein can promote cancer progression, which is associated with poor prognosis and poorer survival benefits for patients.

Although early research results have shown that B7-H3 can stimulate the function of activated T cells, promote the proliferation of CD4 and CD8 cells and the secretion of IFN-γ, continuous in-depth research has shown that B7-H3, as an immune checkpoint, is a negative regulator molecule of T cells, which mainly plays the role of inhibiting T cell function and downregulating T cell activity. Studies by Woong-Kyung Suh and Durbaka V. Prasad have shown that mouse B7-H3 protein can significantly inhibit the proliferation of CD4 and CD8 cells in a dose-dependent manner (<NPL>; <NPL>). Studies by Judith Leitner et al. also have shown that human 4Ig-B7-H3Ig and 2Ig-B7-H3Ig can inhibit the proliferation of T cells in vitro, and inhibit the secretion of related cytokines (IFN-γ, IL-<NUM>, IL-<NUM>, IL-<NUM>) (<NPL>), further analysis has pointed out that B7-H3 mainly inhibits the production of IL-<NUM> to mediate the inhibition of T cell proliferation. Antibodies targeting and neutralizing B7-H3 in mice can significantly promote the progression of experimental autoimmune encephalomyelitis (EAE) and promote the proliferation of CD4 cells, which objectively illustrates the function of B7-H3 in inhibiting T cells in vivo (<NPL>). In Woong-Kyung Suh's study, B7-H3-deficient mice also showed the earlier occurrence of experimental autoimmune encephalomyelitis (caused by Th1 cells) than wild-type mice under immune EAE conditions, which indicates that B7-H3 mainly inhibits Th1 cells (<NPL>). As mentioned above, there is controversy about the function of B7-H3 on T cells, but the function for T cell promotion by B7-H3 has only appeared in mice studies, and there is no yet report on the promotion of T cell function by human B7-H3. Although the receptors of B7-H3 have not been identified, the main point of view in the academic circle is that B7-H3 is a negative regulator molecule of T cells.

Based on the fact that B7-H3 can inhibit T cell activity and thus mediate tumor cells escape from immune surveillance, it is effective to block the binding of B7-H3 to unknown receptors to mediate T cell activation and inhibit tumor cell activity. For example, the existing clinical results of Enoblituzumab show that it has different degrees of remission for different tumors, and has a good curative effect, but there are still many patients with occurrences of disease progression. Therefore, there is still a large clinical unmet need for the simple development of monoclonal antibodies against B7-H3, and the existing clinical results show that its antitumor effect needs to be further improved.

Antibody-drug conjugate (ADC) is a new generation of antibody-targeted therapeutic drugs, which is mainly used in the treatment of cancer tumors. ADC drugs consist of three parts: a small molecule cytotoxic drug (Drug), an antibody (Antibody), and a linker (Linker) that links the antibody with the cytotoxic drug. The small molecule cytotoxic drug is bound to the antibody protein by chemical coupling. ADC drugs utilize antibodies to specifically recognize and guide small molecule drugs to cancer cell targets expressing cancer-specific antigens, and enter the cancer cells through endocytosis. The linker part is broken under the action of intracellular low pH value environment or lysosomal protease, releasing small molecular cytotoxic drugs, so as to achieve the effect of specifically killing cancer cells without damaging normal tissue cells. Therefore, ADC drugs have the characteristics of the targeted specificity of antibodies and the high toxicity of small molecule toxins to cancer cells at the same time, greatly expanding the effective therapeutic window of the drug. Clinical studies have proven that ADC drugs have high efficacy and are relatively stable in the blood, and can effectively reduce the toxicity of small molecule cytotoxic drugs (chemotherapy drugs) to the circulatory system and healthy tissues, and are currently a hot spot in the development of anticancer drugs internationally.

<NPL>) reports on the chemical modification of linkers to provide stable linker-payloads for the generation of antibody-drug conjugates.

<CIT>, <CIT> and <CIT> disclose B7-H3-targeting ADCs comprising exatecan.

The technical problem to be solved by the present disclosure is to overcome the current deficiency of limited types of antibody-drug conjugates, and the present disclosure provides a B7-H3-targeting antibody-drug conjugate, a preparation method therefor, and a use thereof.

The antibody-drug conjugates provided by the present disclosure have good targeting ability, good inhibitory effect on tumor cells that are positive for B7-H3 expression, and good druggability and high safety. The antibody-drug conjugate has an inhibitory effect on B7-H3, and also has a good inhibitory effect on at least one of NCI-N87, A375, LN-<NUM>, PA-<NUM>, MDA-MB-<NUM>, Calu-<NUM>, and Hs-700T cells.

The present disclosure solves the above technical problem through the following technical solution.

The present disclosure provides an antibody-drug conjugate, a pharmaceutically acceptable salt thereof, a solvate thereof, or a solvate of the pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate has a structure shown in formula I;
<CHM>.

In a preferred embodiment of the present disclosure, certain groups of the antibody-drug conjugate are defined as follows, and the definition of any unmentioned groups is as described in any of the above embodiments (this paragraph is hereinafter referred to as "in a preferred embodiment of the present disclosure"):
the b-terminal of L<NUM> is preferably connected to the sulfhydryl group on the antibody in the form of a thioether. Taking
<CHM>
as an example, the connecting form of
<CHM>
with the cysteine residue in the antibody is
<CHM>.

In a preferred embodiment of the present disclosure, when R<NUM> and R<NUM> are each independently C<NUM>-C<NUM> alkyl, the C<NUM>-C<NUM> alkyl is preferably C<NUM>-C<NUM> alkyl, further preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, most preferably methyl.

In a preferred embodiment of the present disclosure, when R<NUM> and R<NUM> are each independently halogen, the halogen is preferably fluorine, chlorine, bromine, or iodine, further preferably fluorine.

In a preferred embodiment of the present disclosure, when R<NUM> and R<NUM> are each independently C<NUM>-C<NUM> alkyl, the C<NUM>-C<NUM> alkyl is preferably C<NUM>-C<NUM> alkyl, further preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, most preferably ethyl.

In a preferred embodiment of the present disclosure, R<NUM> and R<NUM> are each independently C<NUM>-C<NUM> alkyl.

In a preferred embodiment of the present disclosure, R<NUM> and R<NUM> are each independently halogen.

In a preferred embodiment of the present disclosure, R<NUM> and R<NUM> are ethyl.

In a preferred embodiment of the present disclosure, D is
<CHM>
or
<CHM>.

In a preferred embodiment of the present disclosure, when D is
<CHM>
or
<CHM>
the antibody-drug conjugate can be
<CHM>
or
<CHM>.

In a preferred embodiment of the present disclosure, when R<NUM> is C<NUM>-C<NUM> alkyl substituted by one or more than one -NR<NUM>-<NUM>R<NUM>-<NUM>, the C<NUM>-C<NUM> alkyl is preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, more preferably ethyl.

In a preferred embodiment of the present disclosure, when R<NUM> is C<NUM>-C<NUM> alkyl substituted by more than one -NR<NUM>-<NUM>R<NUM>-<NUM>, the "more than one" is two or three.

In a preferred embodiment of the present disclosure, when R<NUM>-<NUM> and R<NUM>-<NUM> are each independently C<NUM>-C<NUM> alkyl, the C<NUM>-C<NUM> alkyl is preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, more preferably methyl.

In a preferred embodiment of the present disclosure, when R<NUM> is C<NUM>-C<NUM> alkyl substituted by one or more than one -NR<NUM>-<NUM>R<NUM>-<NUM>, the -NR<NUM>-<NUM>R<NUM>-<NUM> is preferably -N(CH<NUM>)<NUM>.

In a preferred embodiment of the present disclosure, when R<NUM> is C<NUM>-C<NUM> alkyl substituted by one -NR<NUM>-<NUM>R<NUM>-<NUM>, the C<NUM>-C<NUM> alkyl substituted by one -NR<NUM>-<NUM>R<NUM>-<NUM> is preferably
<CHM>.

In a preferred embodiment of the present disclosure, when R<NUM> is C<NUM>-C<NUM> alkyl substituted by one or more than one R<NUM>-<NUM>S(O)<NUM>-, the C<NUM>-C<NUM> alkyl is preferably C<NUM>-C<NUM> alkyl, further preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, most preferably ethyl.

In a preferred embodiment of the present disclosure, when R<NUM> is C<NUM>-C<NUM> alkyl substituted by more than one R<NUM>-<NUM>S(O)<NUM>-, the "more than one" is two or three.

In a preferred embodiment of the present disclosure, when R<NUM>-<NUM> is C<NUM>-C<NUM> alkyl, the C<NUM>-C<NUM> alkyl is preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, more preferably methyl.

In a preferred embodiment of the present disclosure, when R<NUM> is C<NUM>-C<NUM> alkyl substituted by one R<NUM>-<NUM>S(O)<NUM>-, the C<NUM>-C<NUM> alkyl substituted by one R<NUM>-<NUM>S(O)<NUM>- is
<CHM>.

In a preferred embodiment of the present disclosure, when R<NUM> is C<NUM>-C<NUM> alkyl, the C<NUM>-C<NUM> alkyl is preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, most preferably methyl.

In a preferred embodiment of the present disclosure, m is an integer (such as <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>) or non-integer, preferably <NUM> to <NUM>, more preferably <NUM> to <NUM>, further preferably <NUM> to <NUM>, still more preferably <NUM> to <NUM>, such as <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>.

In a preferred embodiment of the present disclosure, L<NUM> is preferably one or more than one of the phenylalanine residue, alanine residue, glycine residue, isoleucine residue, leucine residue, proline residue, and valine residue; more preferably one or more than one of the phenylalanine residue, alanine residue, glycine residue, and valine residue; further preferably the valine residue and/or the alanine residue; the "more than one" is preferably two or three; p is preferably <NUM>.

In a preferred embodiment of the present disclosure, (L<NUM>)p is preferably
<CHM>
wherein the g-terminal is connected to the c-terminal of L<NUM> through a carbonyl group.

In a preferred embodiment of the present disclosure, n is preferably <NUM> to <NUM>, such as <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>, further such as <NUM> or <NUM>.

In a preferred embodiment of the present disclosure, when R<NUM>-<NUM>, R<NUM>-<NUM>, and R<NUM>-<NUM> are each independently and preferably C<NUM>-C<NUM> alkyl, the C<NUM>-C<NUM> alkyl is preferably C<NUM>-C<NUM> alkyl, further preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, most preferably methyl.

In a preferred embodiment of the present disclosure, R<NUM> is preferably C<NUM>-C<NUM> alkyl substituted by one or more than one -NR<NUM>-<NUM>R<NUM>-<NUM>, C<NUM>-C<NUM> alkyl substituted by one or more than one R<NUM>-<NUM>S(O)<NUM>-, C<NUM>-C<NUM> alkyl, or C<NUM>-C<NUM> cycloalkyl; more preferably C<NUM>-C<NUM> alkyl substituted by one or more than one -NR<NUM>-<NUM>R<NUM>-<NUM>, C<NUM>-C<NUM> alkyl substituted by one or more than one R<NUM>-<NUM>S(O)<NUM>-, or C<NUM>-C<NUM> alkyl; further preferably C<NUM>-C<NUM> alkyl substituted by one or more than one -NR<NUM>-<NUM>R<NUM>-<NUM>, or C<NUM>-C<NUM> alkyl substituted by one or more than one R<NUM>-<NUM>S(O)<NUM>-; most preferably C<NUM>-C<NUM> alkyl substituted by one or more than one R<NUM>-<NUM>S(O)<NUM>-.

In the present disclosure, when Ab is the B7-H3 antibody or the variant of the B7-H3 antibody, the B7-H3 antibody or the variant of the B7-H3 antibody is a residue of the B7-H3 antibody (a group formed by replacing a hydrogen on one of the sulfhydryl groups in B7-H3 antibody) or a residue of a variant of the B7-H3 antibody (a group formed by replacing a hydrogen on one of the sulfhydryl groups in the variant of the B7-H3 antibody).

In a preferred embodiment of the present disclosure, the compound of formula I is any one of the following schemes:.

In a preferred embodiment of the present disclosure, the antibody-drug conjugate is preferably
<CHM>
<CHM>
or
<CHM>.

In a preferred embodiment of the present disclosure, L<NUM> is preferably
<CHM>.

In a preferred embodiment of the present disclosure, Ab is the B7-H3 antibody; D is
<CHM>
L<NUM> is the valine residue and/or the alanine residue, p is <NUM>, (L<NUM>)p is preferably
<CHM>
R<NUM> is C<NUM>-C<NUM> alkyl substituted by one or more than one -NR<NUM>-<NUM>R<NUM>-<NUM>, C<NUM>-C<NUM> alkyl substituted by one or more than one R<NUM>-<NUM>S(O)<NUM>-, or C<NUM>-C<NUM> alkyl, preferably C<NUM>-C<NUM> alkyl substituted by one or more than one - NR<NUM>-<NUM>R<NUM>-<NUM> or C<NUM>-C<NUM> alkyl substituted by one or more than one R<NUM>-<NUM>S(O)<NUM>-, further preferably C<NUM>-C<NUM> alkyl substituted by one or more than one R<NUM>-<NUM>S(O)<NUM>-; R<NUM>-<NUM>, R<NUM>-<NUM>, and R<NUM>-<NUM> are independently C<NUM>-C<NUM> alkyl, preferably methyl; the C<NUM>-C<NUM> alkyl substituted by one or more than one -NR<NUM>-<NUM>R<NUM>-<NUM> is preferably
<CHM>
the C<NUM>-C<NUM> alkyl substituted by one or more than one R<NUM>-<NUM>S(O)<NUM>- is preferably
<CHM>
L<NUM> is
<CHM>
L<NUM> is
<CHM>.

In a preferred embodiment of the present disclosure, the antibody-drug conjugate is preferably any one of the following compounds:
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
or
<CHM>
wherein, Ab is the B7-H3 antibody or the variant of the B7-H3 antibody, and m is <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>.

In a preferred embodiment of the present disclosure, the antibody-drug conjugate is preferably any one of the following compounds:.

The present disclosure also provides a method for preparing the antibody-drug conjugate, comprising the following step: carrying out a coupling reaction between a compound of formula II and Ab-hydrogen as shown below;
<CHM>
wherein L<NUM>, L<NUM>, L<NUM>, R<NUM>, p, and Ab are defined as above.

In the present disclosure, the conditions and operations of the coupling reaction can be conventional conditions and operations for the coupling reaction in the art.

The present disclosure also provides a pharmaceutical composition, comprising substance X and a pharmaceutically acceptable excipient, wherein the substance X is the antibody-drug conjugate, the pharmaceutically acceptable salt thereof, the solvate thereof, or the solvate of the pharmaceutically acceptable salt thereof.

In the pharmaceutical composition, the substance X may be used in a therapeutically effective amount.

The present disclosure also provides substance X for use in inhibiting B7-H3 protein.

The present disclosure also provides substance X for use in treating and/or preventing a tumor, and the tumor is preferably a B7-H3 positive tumor. The B7-H3 positive tumor is preferably one or more than one of B7-H3 positive lung cancer, ovarian cancer, melanoma, pancreatic cancer, breast cancer, brain glioma, prostate cancer, and gastric cancer.

In some embodiments of the present disclosure, for the lung cancer, the lung cancer cells are NCI-<NUM> cells or Calu-<NUM> cells;.

Unless otherwise indicated, the following terms appearing in the specification and claims of the present disclosure have the following meanings:
The pharmaceutical excipient may be an excipient widely used in the field of pharmaceutical production. The excipient is mainly used to provide a safe, stable, and functional pharmaceutical composition, and can also provide a method for the subject to dissolve the active ingredient at a desired rate after administration, or to facilitate effective absorption of the active ingredient after the subject receives administration of the composition. The pharmaceutical excipient can be an inert filler or provide a certain function, such as stabilizing the overall pH value of the composition or preventing degradation of the active ingredient in the composition. The pharmaceutical excipients may include one or more of the following excipients: buffers, chelating agents, preservatives, solubilizers, stabilizers, vehicles, surfactants, colorants, flavoring agents and sweeteners.

The term "pharmaceutically acceptable" refers to salts, solvents, excipients and the like that are generally non-toxic, safe, and suitable for patient use. The "patient" is preferably a mammal, more preferably a human being.

The term "pharmaceutically acceptable salt" refers to salt prepared from the compound of the present disclosure and a relatively non-toxic and pharmaceutically acceptable acid or alkali. When the compound of the present disclosure contains relatively acidic functional groups, an alkali addition salt can be obtained by contacting a sufficient amount of pharmaceutically acceptable alkali with the neutral form of the compound in a pure solution or an appropriate inert solvent. The pharmaceutically acceptable alkali addition salt includes, but is not limited to, lithium salt, sodium salt, potassium salt, calcium salt, aluminium salt, magnesium salt, zinc salt, bismuth salt, ammonium salt, and diethanolamine salt. When the compound of the present disclosure contains relatively alkaline functional groups, an acid addition salt can be obtained by contacting a sufficient amount of pharmaceutically acceptable acid with the neutral form of the compound in a pure solution or an appropriate inert solvent. The pharmaceutically acceptable acid includes inorganic acid, and the inorganic acid includes, but is not limited to, hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, phosphoric acid, phosphorous acid, sulfuric acid, etc. The pharmaceutically acceptable acid includes organic acid, and the organic acid includes, but is not limited to, acetic acid, propionic acid, oxalic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, trans-butenedioic acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, salicylic acid, tartaric acid, methanesulfonic acid, isonicotinic acid, acidic citric acid, oleic acid, tannic acid, pantothenic acid, bitartrate, ascorbic acid, gentisic acid, fumaric acid, gluconic acid, saccharic acid, formic acid, ethanesulfonic acid, pamoic acid (i.e., <NUM>,<NUM>'-methylene-bis(<NUM>-hydroxy-<NUM>-naphthoic acid)), amino acid (e.g., glutamic acid and arginine), etc. When the compound of the present disclosure contains relatively acidic functional groups and relatively alkaline functional groups, it can be converted into an alkali addition salt or an acid addition salt. For details, see <NPL>), or <NPL>).

The term "solvate" refers to a substance formed by combining the compound of the present disclosure with a stoichiometric or non-stoichiometric amount of solvent. Solvent molecules in the solvate can exist in an ordered or non-ordered arrangements. The solvent includes, but is not limited to, water, methanol, ethanol, etc..

Natural or natural sequence B7-H3 can be isolated from nature or produced by recombinant DNA technology, chemical synthesis, or a combination of the above and similar techniques.

Antibody is interpreted in the broadest sense here, which can specifically bind to the target through at least one antigen recognition region located in the variable region of the immunoglobulin molecule, such as carbohydrate, polynucleotide, fat, polypeptide, etc. Specifically, it includes complete monoclonal antibodies, polyclonal antibodies, bispecific antibodies, and antibody fragments, as long as they have the required biological activity. Variants of antibodies of the present disclosure refer to amino acid sequence variants, and covalent derivatives of natural polypeptides, provided that the biological activity equivalent to that of natural polypeptides is retained. The difference between amino acid sequence mutants and natural amino acid sequences is generally that one or more amino acids in the natural amino acid sequence are substituted or one or more amino acids are deleted and/or inserted in the polypeptide sequence. Deletion mutants include fragments of natural polypeptides and N-terminal and/or C-terminal truncation mutants. The amino acid sequence variant is at least <NUM>% (e.g., <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>%) identical to the natural sequence.

The antibodies of the present disclosure can be prepared using techniques well known in the art, such as hybridoma methods, recombinant DNA techniques, phage display techniques, synthetic techniques, or combinations thereof, or other techniques known in the art.

Description of the term "drug-antibody ratio"(DAR). L-D is a reactive group with the conjugation site on the antibody, L is a linker, D is a cytotoxic agent further coupled to the antibody connected to L. In the present disclosure, D is Dxd. The number of DAR of each antibody finally coupled to D is represented by m or m can also represent the number of D coupled to a single antibody. In some embodiments, m is actually an average value between <NUM> and <NUM>, <NUM> and <NUM>, or <NUM> and <NUM>, or m is an integer of <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>; in some embodiments, m is an average value of <NUM>, <NUM>, <NUM>, or <NUM>; in other embodiments, m is an average value of <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM>.

The linker refers to the direct or indirect connection between the antibody and the drug. The linker can be connected to the mAb in many ways, such as through surface lysine, reductive coupling to oxidized carbohydrates, and through cysteine residues released by reducing interchain disulfide bonds. Many ADC connection systems are known in the art, including connections based on hydrazones, disulfides, and peptides.

The term "treatment" or its equivalent expression, when used for, for example, cancer, refers to a procedure or process for reducing or eliminating the number of cancer cells in a patient or alleviating the symptoms of cancer. The "treatment" of cancer or another proliferative disorder does not necessarily mean that the cancer cells or other disorders will actually be eliminated, but the number of cells or disorders will actually be reduced or the symptoms of cancers or other disorders will actually be alleviated. Generally, the method of treating cancer will be carried out even if it has only a low probability of success, but it is still considered to induce an overall beneficial course of action considering the patient's medical history and estimated survival expectation.

The term "prevention" refers to a reduced risk of acquiring or developing a disease or disorder.

The term "cycloalkyl" refers to a saturated cyclic hydrocarbon group with three to twenty carbon atoms (e.g., C<NUM>-C<NUM> cycloalkyl), including monocyclic cycloalkyl. The cycloalkyl contains <NUM> to <NUM> carbon atoms, preferably <NUM> to <NUM> carbon atoms, and more preferably <NUM> to <NUM> carbon atoms. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, and cyclodecyl.

The term "alkyl" refers to a straight or branched chain alkyl group with a specified number of carbon atoms. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, isopentyl, n-hexyl, n-heptyl, n-octyl, and similar alkyl groups.

The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

The term "heteroaryl" refers to an aryl group (or aromatic ring) containing <NUM>, <NUM>, <NUM>, or <NUM> heteroatoms independently selected from N, O, and S, which may be a monocyclic aromatic system, such as furyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, thiophenyl, isoxazolyl, oxazolyl, diazolyl, imidazolyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, and isothiazolyl.

The term "aryl" refers to any stable monocyclic or bicyclic carbocycle, wherein all rings are aromatic rings. Examples of the aryl moiety include phenyl or naphthyl.

The above preferred conditions can be combined arbitrarily to obtain preferred embodiments of the present disclosure without violating common knowledge in the art.

Unless otherwise specified, room temperature in the present disclosure refers to <NUM> to <NUM>.

The reagents and raw materials used in the present disclosure are all commercially available.

The positive progressive effects of the present disclosure are as follows:.

<FIG> shows the construction of expression vectors for the light and heavy chains of an FDA016 antibody; wherein Ab-L is the light chain of the antibody, and Ab-H is the heavy chain of the antibody.

The present disclosure will be further described below with reference to examples, but the present disclosure is not therefore limited to the scope of the examples. Experimental methods without specific conditions in the following examples are selected according to conventional methods and conditions, or according to the commercial specification.

In the present disclosure, the monoclonal antibody FDA016 with high affinity and specific targeting B7-H3 was selected, the amino acid sequence of its light chain was shown in SEQ ID NO: <NUM>, and the amino acid sequence of its heavy chain was shown in SEQ ID NO: <NUM>. The light and heavy chain nucleotide sequences of FDA016 were obtained by whole gene synthesis (Suzhou Genewiz). They were separately constructed into the pV81 vector (as shown in <FIG>) by double digestion with EcoR I and Hind III (purchased from TAKARA), and then transformed into Trans <NUM>-T1 competent cells (purchased from Beijing TransGen Biotech, product number: CD501-<NUM>) by ligation, which were picked for cloning, PCR identification and sent for sequencing confirmation. Positive clones were cultured and expanded for plasmid extraction, thus obtaining the antibody light chain eukaryotic expression plasmid FDA016-L/pV81 and the antibody heavy chain eukaryotic expression plasmid FDA016-H/pV81. These two plasmids were linearized by digestion with XbaI (purchased from Takara, product number: <NUM>). The light and heavy chain eukaryotic expression plasmids were transformed into CHO cells adapted to suspension growth (purchased from ATCC) at a ratio of <NUM>/<NUM> by electroporation. After electroporation, the cells were seeded at <NUM> to <NUM> cells/well in a <NUM>-well plate. After <NUM> weeks of culture, the expression level was measured by HTRF method (homogeneous time-resolved fluorescence). The top ten cell pools in terms of expression level were selected for expansion and cryopreservation. A cell was revived into a <NUM> shake flask (culture volume of <NUM>) and cultured at <NUM>, <NUM>% CO<NUM>, and <NUM> rpm by vibration for <NUM> days, then expanded to a <NUM> shake flask (culture volume of <NUM>) and cultured at <NUM>, <NUM>% CO<NUM>, and <NUM> rpm by vibration. Starting on the 4th day, <NUM> to <NUM>% of the initial culture volume of replenishment culture medium was added every other day. The culture was ended on 10th to 12th day and the harvest liquid was centrifuged at <NUM> rpm for <NUM> minutes to remove the cell precipitate. The supernatant was collected and filtered through a <NUM> filter membrane. The treated sample was purified using a MabSelect affinity chromatography column (purchased from GE) to obtain antibody FDA016.

The amino acid sequence of the FDA016 light chain is shown below:
<IMG>
<IMG>.

The amino acid sequence of FDA016 heavy chain is shown below:
<IMG>.

(S)-<NUM>-Azidopropionic acid (<NUM>, <NUM> mmol) and <NUM>-aminobenzyl alcohol (<NUM>, <NUM> mmol) were dissolved in a mixed solvent of <NUM> of dichloromethane and methanol (in a volume ratio of <NUM>:<NUM>), and then <NUM>-ethoxy-<NUM>-ethoxycarbonyl-<NUM>,<NUM>-dihydroquinoline (<NUM>, <NUM> mmol) was added thereto. The reaction was carried out at room temperature for <NUM> hours. The solvent was then evaporated under reduced pressure and the obtained crude product was purified by silica gel column chromatography [dichloromethane: ethyl acetate = <NUM>:<NUM> (v/v)] to obtain intermediate <NUM> (<NUM>, yield of <NUM>%), ESI-MS m/z: <NUM> (M+H).

Intermediate <NUM> (<NUM>, <NUM> mmol) was mixed with bis(p-nitrophenyl)carbonate (<NUM>, <NUM> mmol) and dissolved in <NUM> of anhydrous N,N-dimethylformamide, and then <NUM> of triethylamine was added thereto, and the reaction was carried out at room temperature for <NUM> hours. After the complete reaction of the raw materials was monitored by liquid chromatography-mass spectrometry, methylamine hydrochloride (<NUM>, <NUM> mmol) was added thereto, and the reaction was continued at room temperature for <NUM> hour. After the reaction was completed, most of the solvent was removed by distillation under reduced pressure, then <NUM> of water and <NUM> of ethyl acetate were added thereto. The organic phase was collected after the phases were separated, and the organic phase was dried and concentrated, and then the obtained crude product was purified by silica gel column chromatography [dichloromethane: ethyl acetate = <NUM>:<NUM> (v/v)] to obtain intermediate <NUM> (<NUM>, yield of <NUM>%), ESI-MS m/z: <NUM> (M+H).

Intermediate <NUM> (<NUM>, <NUM> mmol) was mixed with polyformaldehyde (<NUM>, <NUM> mmol) and dissolved in <NUM> of anhydrous dichloromethane. Trimethylchlorosilane (<NUM>, <NUM> mmol) was slowly added thereto and the reaction was carried out at room temperature for <NUM> hours to obtain a crude solution of intermediate <NUM>. The reaction was monitored by liquid chromatography-mass spectrometry after sampling and quenching with methanol. After the reaction was completed, the reaction mixture was filtered and then tert-butyl hydroxyacetate (<NUM>, <NUM> mmol) and triethylamine (<NUM>, <NUM> mmol) were added to the filtrate and the reaction was continued at room temperature for <NUM> hours. After the reaction was completed, most of the solvent was removed by distillation under reduced pressure, and then the obtained crude product was purified by silica gel column chromatography [petroleum ether: ethyl acetate = <NUM>:<NUM> (v/v)] to obtain intermediate <NUM> (<NUM>, yield of <NUM>%), ESI-MS m/z: <NUM> (M+H).

Intermediate <NUM> (<NUM>, <NUM> mmol) was dissolved in <NUM> of anhydrous tetrahydrofuran, and <NUM> of water was added thereto, and then tris(<NUM>-carboxyethylphosphine) hydrochloride (<NUM>, <NUM> mmol) was added thereto and the reaction was carried out for <NUM> hours at room temperature. After the reaction was completed, the tetrahydrofuran was removed by distillation under reduced pressure, and then the mixture was extracted with ethyl acetate. The obtained organic phase was dried and evaporated to remove the solvent under reduced pressure, and purified by silica gel column chromatography [dichloromethane: methanol = <NUM>:<NUM> (v/v)] to obtain intermediate <NUM> (<NUM>, yield of <NUM>%), ESI-MS m/z: <NUM> (M+H).

Intermediate <NUM> (<NUM>, <NUM> mmol) was dissolved in <NUM> of a mixed solvent of dichloromethane and methanol (v/v = <NUM>:<NUM>), and <NUM> of trifluoroacetic acid was slowly added thereto, and the reaction was carried out at room temperature for <NUM>. After the reaction was completed, an equal volume of water and ethyl acetate were added thereto, and the organic phase was dried and concentrated, and the obtained crude product was directly used in the next step.

The crude product obtained from the previous step was dissolved in <NUM> of anhydrous N,N-dimethylformamide, and then Fmoc-L-valine hydroxysuccinimide ester (<NUM>, <NUM> mmol) and triethylamine (<NUM>) were added thereto, and the reaction was carried out at room temperature for <NUM> hours. After the reaction was completed, most of the solvent was removed by distillation under reduced pressure, and then the obtained crude product was purified by silica gel column chromatography [dichloromethane: methanol = <NUM>:<NUM> (v/v)] to obtain intermediate <NUM> (<NUM>, yield of <NUM>%), ESI-MS m/z: <NUM> (M+H).

Intermediate <NUM> (<NUM>, <NUM> mmol) was mixed with Exatecan methanesulfonate (<NUM>, <NUM> mmol) in <NUM> of anhydrous N,N-dimethylformamide, and then <NUM>-(<NUM>-azabenzotriazol-<NUM>-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (<NUM>, <NUM> mmol) and <NUM> of triethylamine were added thereto, and the reaction was carried out at room temperature for <NUM> hours. After the reaction was completed, the solvent was removed by distillation under reduced pressure, and then the obtained crude product was purified by silica gel column chromatography [chloroform: methanol = <NUM>:<NUM> (v/v)] to obtain intermediate <NUM> (<NUM>, yield of <NUM>%), ESI-MS m/z: <NUM> (M+H).

Intermediate <NUM> (<NUM>, <NUM> mmol) was dissolved in <NUM> of anhydrous DMF, then <NUM> of <NUM>,<NUM>-diazabicyclo[<NUM>. <NUM>]undec-<NUM>-ene was added thereto, and the reaction was carried out at room temperature for <NUM> hour. After the reaction of the raw materials was completed, N-succinimidyl <NUM>-maleimidohexanoate (<NUM>, <NUM> mmol) was added directly, and the reaction mixture was stirred at room temperature for <NUM> hour. The solvent was removed by distillation under reduced pressure, and then the obtained crude product was purified by silica gel column chromatography [chloroform: methanol = <NUM>:<NUM> (v/v)] to obtain the title compound (<NUM>, yield of <NUM>%), ESI-MS m/z: <NUM> (M+H).

Commercially available intermediate <NUM> (<NUM>, <NUM> mmol) was mixed with paraformaldehyde (<NUM>, <NUM> mmol) and dissolved in <NUM> of anhydrous dichloromethane. Then, trimethylchlorosilane (<NUM>, <NUM> mmol) was added slowly. After the addition was completed, the reaction was carried out at room temperature for <NUM> hours. Then, the reaction was monitored by liquid chromatography-mass spectrometry after sampling and quenching with methanol. After the reaction was completed, the reaction mixture was filtered, and then tert-butyl <NUM>-hydroxyacetate (<NUM>, <NUM> mmol) and pempidine (<NUM>) were added to the filtrate, and the reaction was continued at room temperature for about <NUM> hours. After the reaction was completed, most of the solvent was removed by distillation under reduced pressure, and the obtained crude product was purified by silica gel column chromatography [dichloromethane: methanol = <NUM>:<NUM> (v/v)] to obtain intermediate <NUM> (<NUM>, yield of <NUM>%), ESI-MS m/z: <NUM> (M+H).

The crude product obtained from the previous step was mixed with Exatecan methanesulfonate (<NUM>, <NUM> mmol) in <NUM> of anhydrous N,N-dimethylformamide, and then <NUM>-(<NUM>-azabenzotriazol-<NUM>-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (<NUM>, <NUM> mmol) and <NUM> of triethylamine were added thereto, and the reaction was carried out at room temperature for <NUM> hours. After the reaction was completed, the solvent was removed by distillation under reduced pressure, and then the obtained crude product was purified by silica gel column chromatography [chloroform: methanol = <NUM>:<NUM> (v/v)] to obtain intermediate <NUM> (<NUM>, <NUM>%), ESI-MS m/z: <NUM> (M+H).

Intermediate <NUM> (<NUM>, <NUM> mmol) was dissolved in <NUM> of anhydrous tetrahydrofuran, and <NUM> of water was added thereto, then <NUM> of <NUM> mol/L triethylphosphine aqueous solution was added thereto, and the reaction was carried out at room temperature for <NUM> hours. After the reaction was monitored to be completed, the reaction mixture was distilled under reduced pressure to remove tetrahydrofuran. Sodium bicarbonate was added to the remaining aqueous solution to adjust the pH to neutral, and then dichloromethane was added for extraction. The obtained organic phase was dried and evaporated under reduced pressure to remove the solvent. The obtained crude product was purified by silica gel column chromatography [dichloromethane: methanol = <NUM>:<NUM> (v/v)] to obtain intermediate <NUM> (<NUM>, yield of <NUM>%), ESI-MS m/z: <NUM> (M+H).

Intermediate <NUM> (<NUM>, <NUM> mmol) obtained according to the previous synthesis method was mixed with the commercially available raw material MC-V(<NUM>, <NUM> mmol) in <NUM> of dichloromethane, and the condensation agent <NUM>-ethoxy-<NUM>-ethoxycarbonyl-<NUM>,<NUM>-dihydroquinoline (<NUM>, <NUM> mmol) was added to react overnight at room temperature. After the reaction was completed, the solvent was evaporated under reduced pressure and the obtained crude product was purified by silica gel column chromatography [dichloromethane: methanol = <NUM>:<NUM> (v/v)] to obtain compound LE13 (<NUM>, yield of <NUM>%), ESI-MS m/z: <NUM> (M+H).

Commercially available intermediate <NUM> (<NUM>, <NUM> mmol) was mixed with polyformaldehyde (<NUM>, <NUM> mmol) and dissolved in <NUM> anhydrous dichloromethane. Then, trimethylchlorosilane (<NUM>, <NUM> mmol) was slowly added thereto, and the reaction was carried out at room temperature for <NUM> hours. The reaction was monitored by liquid chromatography-mass spectrometry after sampling and quenching with methanol. After the reaction was completed, the reaction mixture was filtered and then tert-butyl <NUM>-hydroxyacetate (<NUM>, <NUM> mmol) and triethylamine (<NUM>, <NUM> mmol) were added to the filtrate. The reaction was continued at room temperature for about <NUM> hours. After the reaction was completed, most of the solvent was removed by distillation under reduced pressure and the obtained crude product was purified by silica gel column chromatography [dichloromethane: methanol = <NUM>:<NUM> (v/v)] to obtain intermediate <NUM> (<NUM>, yield of <NUM>%), ESI-MS m/z: <NUM> (M+H), <NUM>H NMR (<NUM>, CDCl<NUM>) δ <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (q, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (s, <NUM>).

The obtained crude product was mixed with Exatecan methanesulfonate (<NUM>, <NUM> mmol) in <NUM> of anhydrous N,N-dimethylformamide, and then <NUM>-(<NUM>-azabenzotriazol-<NUM>-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (<NUM>, <NUM> mmol) and <NUM> of triethylamine were added thereto, and the reaction was carried out at room temperature for <NUM> hours. After the reaction was completed, the solvent was removed by distillation under reduced pressure, and then the obtained crude product was purified by silica gel column chromatography [dichloromethane: methanol = <NUM>:<NUM> (v/v)] to obtain intermediate <NUM> (<NUM>, yield of <NUM>%), ESI-MS m/z: <NUM> (M+H). <NUM>H NMR (<NUM>, CDCl<NUM>) δ <NUM> (d, J = <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>).

Intermediate <NUM> (<NUM>, <NUM> mmol) was dissolved in <NUM> of anhydrous tetrahydrofuran, and <NUM> of water was added thereto, then <NUM> of <NUM> mol/L triethylphosphine aqueous solution was added thereto, and the reaction was carried out at room temperature for <NUM> hours. After the reaction was completed, the reaction mixture was distilled under reduced pressure to remove tetrahydrofuran. Sodium bicarbonate was added to the remaining aqueous solution to adjust the pH to neutral, and then dichloromethane was added for extraction. The obtained organic phase was dried and evaporated under reduced pressure to remove the solvent. The obtained crude product was purified by silica gel column chromatography [dichloromethane: methanol = <NUM>:<NUM> (v/v)] to obtain intermediate <NUM> (<NUM>, yield of <NUM>%), ESI-MS m/z: <NUM> (M+H). <NUM>H NMR (<NUM>, DMSO) δ <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m,<NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>).

Intermediate <NUM> (<NUM>, <NUM> mmol) was mixed with the commercially available raw material MC-V (<NUM>, <NUM> mmol) in <NUM> of dichloromethane, and the condensation agent <NUM>-ethoxy-<NUM>-ethoxycarbonyl-<NUM>,<NUM>-dihydroquinoline (<NUM>, <NUM> mmol) was added to react overnight at room temperature. After the reaction was completed, the solvent was evaporated under reduced pressure and the obtained crude product was purified by silica gel column chromatography [dichloromethane: methanol=<NUM>:<NUM> (v/v)] to obtain compound LE14 (<NUM>, yield of <NUM>%), ESI-MS m/z: <NUM> (M+H). <NUM>H NMR (<NUM>, DMSO) δ <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>).

Intermediate VI could be prepared by replacing the methylamine hydrochloride in step b with the corresponding commercially available amino compound, using Fmoc-L-valyl-L-alanine as the starting material and referring to steps a and b in the synthesis method of intermediate <NUM> in Example <NUM>-<NUM>. The subsequent steps were carried out starting from intermediate VI, and with the same methods as those in steps c, d, f, and h of Example <NUM>-<NUM>, and intermediate IX similar to intermediate <NUM> was obtained. Then, following the same steps i and j as Example <NUM>, the amino protecting group was removed, and condensed with different commercially available maleimide compounds to obtain the final product. The structures of the amino compounds and maleimides used are shown in Table <NUM>. Compound LE15: graywhite solid, ESI-MS m/z: <NUM> (M+H); compound LE16: light yellow solid, ESI-MS m/z: <NUM> (M+H); compound LE17: yellow solid, ESI-MS m/z: <NUM> (M+H); compound LE18: light yellow solid, ESI-MS m/z: <NUM> (M+H); compound LE19: light yellow solid, ESI-MS m/z: <NUM> (M+H); compound LE20: light yellow solid, ESI-MS m/z: <NUM> (M+H).

Commercially available Exatecan methanesulfonate (<NUM>, <NUM> mmol) was mixed with commercially available <NUM>-(tert-butyldimethylsilyloxy)acetic acid (CAS: <NUM>-<NUM>-<NUM>, <NUM>, <NUM> mmol) in <NUM> anhydrous dichloromethane. The condensation agent HATU (<NUM>, <NUM> mmol) and <NUM> of pyridine were added thereto and stirred at room temperature for <NUM> hours. After the reaction was completed, the solvent was evaporated to dryness under reduced pressure, and the obtained crude product was purified by column chromatography [dichloromethane: methanol = <NUM>:<NUM> (v/v)] to obtain title compound DXD-<NUM> (<NUM>, yield of <NUM>%), ESI-MS m/z: <NUM> (M+H). <NUM>H NMR (<NUM>, CDCl<NUM>) δ <NUM> (d, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>).

Intermediate V could be prepared by replacing the methylamine hydrochloride in step b with the corresponding commercially available amino compound, referring to the preparation method of compound <NUM> in Example <NUM>-<NUM>.

Intermediate V was reacted with DXD-<NUM>, and then treated with <NUM>% trifluoroacetic acid/dichloromethane solution to obtain intermediate X. Then, intermediate X was reacted according to the subsequent steps e, g, i, and j of compound <NUM> in Example <NUM>-<NUM>: Intermediate X was reduced to obtain an amino compound, and the amino compound was then condensed with Fmoc-L-valine hydroxysuccinimide ester. Then the Fmoc protecting group of the amino group was removed from the obtained product, and the obtained amino product was then reacted with N-succinimidyl <NUM>-maleimidohexanoate to obtain the final product. Compound LE21: yellow solid, ESI-MS m/z: <NUM> (M+H); compound LE22: yellow solid, ESI-MS m/z: <NUM> (M+H). <CHM>
<CHM>.

Referring to the synthesis method of LE15 in Examples <NUM> to <NUM>, SN-<NUM> (<NUM>-ethyl-<NUM>-hydroxycamptothecin) was reacted with intermediate VII (R<NUM> is methylsulfonyl ethyl) to obtain compound LS13 after deprotection, condensation and other steps: <NUM>H NMR (<NUM>, DMSO) δ <NUM> (d, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>).

Compound GGFG-Dxd was prepared according to the known synthesis method reported in <CIT>. ESI-MS m/z: <NUM> (M+H), <NUM>H-NMR (<NUM>, DMSO-d<NUM>) δ <NUM> (t, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (dt, J = <NUM>, <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (dd, J= <NUM>, <NUM>, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, <NUM>), <NUM> (t, J = <NUM>, <NUM>).

The antibody FDA016 against B7-H3 was prepared according to the method of Example <NUM> and was replaced into <NUM> PB/<NUM> EDTA buffer (pH <NUM>) using a G25 desalting column. <NUM> equivalents of TECP were added thereto and the mixture was stirred at <NUM> for <NUM> hours to fully open the disulfide bonds between the antibody chains. Then, phosphoric acid was used to adjust the pH of the reduced antibody solution to <NUM> and the temperature of the water bath was lowered to <NUM> for coupling reaction. The linker-drug conjugates LE12 to LE22, LS13, and GGFG-Dxd prepared according to the above Example <NUM> were dissolved in DMSO respectively and <NUM> equivalents of linker-drug conjugate were added dropwise to the reduced antibody solution. Additional DMSO was added to a final concentration of <NUM>% (v/v) and the reaction was stirred at <NUM> for <NUM> hours. After the reaction was completed, the sample was filtered through a <NUM> membrane. The tangential flow filtration system was used to purify and remove unconjugated small molecules. The buffer was a <NUM> PB/<NUM> EDTA solution (pH <NUM>). After purification, a final concentration of <NUM>% sucrose was added and stored in a -<NUM> refrigerator. The absorbance values were measured at <NUM> and <NUM> by UV method, respectively, and the DAR value was calculated.

The coupling reaction was carried out in the same manner as in this example and all samples were prepared according to the highest DAR (i.e., excessive coupling). The occurrence of precipitation during each coupling reaction was observed and the polymer ratio and recovery rate after each coupling reaction were calculated. The results are also shown in Table <NUM>.

In practical research, it was found that the linker-drug conjugate GGFG-Dxd also produced precipitation when coupled with other antibodies and had a high aggregation ratio, which lacked universality. However, when most of the linker-drug conjugates of the present technical solution were attempted to be coupled with different antibodies including F016, no precipitation was produced and the aggregation ratio was within the normal range, indicating that the antibody-drug conjugates provided by the present disclosure have good solubility and druggability, and that no precipitation during the coupling process is also very conducive to the preparation of antibody-drug conjugates.

Calu-<NUM> (ATCC) cells were selected as the cell line for in vitro activity detection. <NUM> cells per well were seeded in a <NUM>-well cell culture plate and cultured for <NUM> to <NUM> hours. The antibody-drug conjugates prepared according to the method of Example <NUM> were formulated into test solutions with <NUM> concentration gradients of <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> using L15 cell culture medium containing <NUM>% FBS. The diluted test solutions were added to the culture plate containing the seeded cells at <NUM>µL/well and incubated for <NUM> hours at <NUM> in a <NUM>% CO<NUM> incubator. CellTiter-Glo® Luminescent Cell Viability Assay Reagent (promega, G9243) (<NUM>µL/well) was added and the plate was shaken at <NUM> rpm at room temperature for <NUM> minutes to mix well. The data were read using a SpectraMaxL microplate reader (OD <NUM>, reading at <NUM> intervals) and the IC50 results were calculated as shown in Table <NUM>.

Using the same method as above, the cytotoxic activity of each antibody-drug conjugate against multiple tumor cells of NCI-N87, A375, Hs-700T, LN-<NUM>, MDA-MB-<NUM>, PA-<NUM>, and Raji purchased from ATCC was tested. The results are shown in Table <NUM>. From the results in Table <NUM>, it can be seen that the antibody-drug conjugates provided by the present disclosure have excellent in vitro killing activity against cells such as Calu-<NUM>, NCI-N87, A375, Hs-700T, LN-<NUM>, MDA-MB-<NUM>, and PA-<NUM>. However, the antibody-drug conjugate has no killing activity on Raji-negative cells, indicating that the prepared ADC has specific targeted killing activity.

This example evaluates the stability of the antibody-drug conjugate prepared according to the method of Example <NUM> in human plasma. Specifically, in this example, the antibody-drug conjugate of Example <NUM> was added to human plasma and placed in a <NUM> water bath for <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> days. An internal standard (Exatecan as an internal standard substance) was added and extracted and then detected by high-performance liquid chromatography to detect the release of free drugs. The results are shown in Table <NUM>.

The plasma stability results show that the antibody-drug conjugate provided by the present disclosure has good plasma stability.

The linker-drug conjugate (LE14 and GGFG-Dxd) was co-incubated with cathepsin B in three different pH (<NUM>, <NUM>, <NUM>) buffers. Samples were taken at different time points and entered into a high-performance liquid chromatography-mass spectrometry instrument. The external standard method (with DXD as the external standard) was used to determine the release percentage of the drug. The experimental results (as shown in Table <NUM>) show that GGFG-Dxd has a slow speed of enzyme digestion within the pH range used, while the Linker-drug conjugate LE14 employed by the present disclosure can be quickly enzymatically digested within the pH range of <NUM> to <NUM>.

NCI-N87 cell line was selected as the experimental cell lines. After the sample was incubated in cathepsin B system (<NUM> sodium acetate-acetic acid buffer, <NUM> dithiothreitol, pH <NUM>) at <NUM> for <NUM> hours, the obtained sample was diluted with culture medium to different concentrations. <NUM> concentrations (<NUM> to <NUM>-fold dilution) were set from <NUM> to <NUM> of SN-<NUM> concentration. The killing (inhibitory) ability of the cell line was observed for <NUM> hours. The IC50 value was calculated by reading the fluorescence data after chemical luminescent staining with CellTiter-Glo® Luminescent Cell Viability Assay.

The above enzyme digestion samples obtained by incubating in a cathepsin B system at <NUM> for <NUM> hours were precipitated with an appropriate amount of ethanol to remove protein and detected by high-performance liquid chromatography to release small molecule compounds. The <NUM>-hour release rate was measured with an equal amount of SN-<NUM> as a reference, and the results showed that the release rate reached <NUM>%.

The experimental results (as shown in Table <NUM>) show that after enzyme digestion treatment, the cytotoxic activity of FDA016-LS13 is almost the same as that of SN-<NUM> at an equivalent dose, which also indicates that FDA016-LS13 has almost completely released SN-<NUM> under the action of cathepsin B and played a role. However, FDA016-LS13 may have undergone unpredictable changes when it is endocytosed into lysosomes, resulting in SN-<NUM> not being able to function effectively.

<NUM>- to <NUM>-week-old female Balb/c nude mice were subcutaneously injected with <NUM>×<NUM><NUM> human lung cancer cells (Calu-<NUM>) dissolved in <NUM>µL of PBS solution on the right side of the neck and back. When the tumor grew to an average volume of <NUM> to <NUM><NUM>, mice were randomly divided into <NUM> groups according to tumor size and mouse weight, with <NUM> animals in each group. The groups were blank control group, <NUM>/kg FDA016-GGFG-Dxd group, <NUM>/kg FDA016-GGFG-Dxd group, <NUM>/kg FDA016 -LE14 group, and <NUM>/kg FDA016-LE14 group, respectively, administered intraperitoneally once a week. The animal weight and tumor volume were measured twice a week, and the survival status of the experimental animals was observed during the experiment process. As shown in Table <NUM>, the average tumor volume of the mice in the blank control group was <NUM><NUM> at the end of treatment. The average tumor volume of the FDA016-GGFG-Dxd treatment group at <NUM>/kg was <NUM><NUM> on the 14th day after the end of treatment, and the average tumor volume of the FDA016-GGFG-Dxd treatment group at <NUM>/kg was <NUM><NUM> on the 14th day after the end of treatment. The average tumor volume of the FDA016-LE14 treatment group at <NUM>/kg was <NUM><NUM> on the 14th day after the end of treatment, and the average tumor volume of the FDA016-LE14 treatment group at <NUM>/kg was <NUM><NUM> on the 14th day after the end of treatment. The experimental results show that FDA016-LE14 has good in vivo antitumor activity, and all experimental mice have no death or weight loss, indicating that FDA016-LE14 has good safety.

Claim 1:
An antibody-drug conjugate, a pharmaceutically acceptable salt thereof, a solvate thereof, or a solvate of the pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate has a structure shown in formula I;
<CHM>
wherein Ab is a B7-H3 antibody or a variant of the B7-H3 antibody; m is <NUM> to <NUM>;
the amino acid sequence of the light chain in the B7-H3 antibody is shown in SEQ ID NO: <NUM>, and the amino acid sequence of the heavy chain is shown in SEQ ID NO: <NUM>;
the variant of the B7-H3 antibody is at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>% identical to the B7-H3 antibody;
D is a cytotoxic drug topoisomerase inhibitor, wherein the cytotoxic drug topoisomerase inhibitor is
<CHM>
R<NUM> and R<NUM> are each independently H, C<NUM>-C<NUM> alkyl, or halogen; R<NUM> and R<NUM> are each independently H, C<NUM>-C<NUM> alkyl, or halogen; R<NUM> and R<NUM> are each independently C<NUM>-C<NUM> alkyl;
R<NUM> is C<NUM>-C<NUM> alkyl substituted by one or more than one -NR<NUM>-<NUM>R<NUM>-<NUM>, C<NUM>-C<NUM> alkyl substituted by one or more than one R<NUM>-<NUM>S(O)<NUM>-, C<NUM>-C<NUM> alkyl, C<NUM>-C<NUM> cycloalkyl, C<NUM>-C<NUM> aryl, or <NUM>- to <NUM>-membered heteroaryl; the heteroatom in the <NUM>- to <NUM>-membered heteroaryl is selected from one or more than one of N, O, and S, and the number of heteroatoms is <NUM>, <NUM>, <NUM>, or <NUM>; the R<NUM>-<NUM>, R<NUM>-<NUM>, and R<NUM>-<NUM> are each independently C<NUM>-C<NUM> alkyl;
L<NUM> is independently one or more than one of a phenylalanine residue, alanine residue, glycine residue, glutamic acid residue, aspartic acid residue, cysteine residue, histidine residue, isoleucine residue, leucine residue, lysine residue, methionine residue, proline residue, serine residue, threonine residue, tryptophan residue, tyrosine residue, and valine residue; p is <NUM> to <NUM>;
L<NUM> is
<CHM>
<CHM>
or
<CHM>
wherein n is independently <NUM> to <NUM>, the c-terminal is connected to L<NUM> through a carbonyl group, and the f-terminal is connected to the d-terminal of L<NUM>;
L<NUM> is
<CHM>
wherein the b-terminal is connected to the Ab, and the d-terminal is connected to the f-terminal of L<NUM>.