Pharmaceutical composition of Anti-ROR1 antibody-drug conjugate and use thereof

Abstract

Provided is a pharmaceutical composition of an anti-ROR1 antibody-drug conjugate. The pharmaceutical composition comprises an anti-ROR1 antibody-drug conjugate, a buffer, a stabilizer, and a surfactant. The anti-ROR1 antibody comprises a first antigen-binding domain and a second antigen-binding domain. The drug is Eribulin, which is linked to the anti-ROR1 antibody by means of a linker. By optimizing the component ratio and the preparation process, the pharmaceutical composition of the anti-ROR1 antibody-drug conjugate exhibits excellent physical and chemical stability, ensuring that the anti-ROR1 antibody-drug conjugate can maintain a stable effect under long-term storage conditions.

Description

A pharmaceutical composition of an anti-ROR1 antibody-drug conjugate and its application Technical Field

[0001] This disclosure pertains to the pharmaceutical field and specifically relates to pharmaceutical compositions of anti-ROR1 antibody-drug conjugates and their applications. Background Technology

[0002] ROR1 (Receptor tyrosine kinase-like Orphan Receptor 1) is a transmembrane receptor tyrosine kinase belonging to the receptor tyrosine kinase (RTK) superfamily. Researchers initially discovered ROR1 during nervous system development, but its expression level is low in normal adult tissues. However, studies have found that ROR1 is highly expressed in various malignant tumors and is closely associated with tumor invasion, metastasis, and poor prognosis.

[0003] The ROR1 protein comprises an extracellular region (containing an immunoglobulin-like domain, a cysteine-rich domain, and a Kringle domain), a transmembrane region, and an intracellular region (containing a tyrosine kinase domain and a proline-rich domain). This unique structure enables ROR1 to participate in the regulation of multiple cellular signaling pathways.

[0004] Researchers have found high expression of ROR1 in various cancers, including chronic lymphocytic leukemia (CLL), breast cancer, lung cancer, and ovarian cancer. Studies have shown that ROR1 promotes tumor cell proliferation, survival, and metastasis by activating multiple downstream signaling pathways, such as the PI3K / AKT, MAPK, and Wnt signaling pathways.

[0005] Of particular note is the high expression level of ROR1 in tumor stem cells, suggesting that ROR1 may play a crucial role in tumor self-renewal and drug resistance. Furthermore, researchers found that ROR1 is involved in the epithelial-mesenchymal transition (EMT) process in tumor cells, a key mechanism of tumor metastasis.

[0006] Because ROR1 is expressed at low levels in normal adult tissues but highly in various tumors, it has become a highly attractive anti-tumor target. Currently, researchers are developing various therapeutic strategies targeting ROR1, including monoclonal antibodies, bispecific antibodies, antibody-drug conjugates (ADCs), and chimeric antigen receptor T-cell (CAR-T) therapy.

[0007] However, the development of antibody-drug conjugates (ADCs) faces stability challenges. ADCs can undergo various chemical and physical degradations, particularly the higher-order structures of the antibody moiety, which are prone to denaturation, aggregation, and precipitation. These unstable products can affect the safety and efficacy of the drug. Researchers consider polymer formation a critical quality attribute (CQA) for biopharmaceutical safety, directly impacting the safety of anti-ROR1 ADCs. Obtaining high-purity products during manufacturing is crucial, as is ensuring structural stability during transportation, storage, and use, which is especially important for patients requiring long-term use of anti-ROR1 ADCs.

[0008] Therefore, it remains essential to develop a novel anti-ROR1 antibody-drug conjugate formulation to improve its stability, thereby enhancing the uniformity and consistency of product quality and improving its stability in use. Summary of the Invention

[0009] Through repeated experimental research, the inventors conducted extensive screening of the components and contents of various buffer systems and discovered a highly stable formulation containing an anti-ROR1 antibody-drug conjugate, which has a very broad market application prospect.

[0010] The first aspect of this disclosure discloses a pharmaceutical composition comprising an anti-ROR1 antibody-drug conjugate, a buffer, a stabilizer, and a surfactant; wherein the anti-ROR1 antibody comprises a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain comprises a heavy chain variable region VH1 and a light chain variable region VL1, and the second antigen-binding domain comprises a heavy chain variable region VH2 and a light chain variable region VL2, wherein:

[0011] The heavy chain variable region VH1 includes HCDR1 as shown in SEQ ID NO:17, HCDR2 as shown in SEQ ID NO:32, and HCDR3 as shown in SEQ ID NO:19, and the light chain variable region VL1 includes LCDR1 as shown in SEQ ID NO:20, LCDR2 as shown in SEQ ID NO:21, and LCDR3 as shown in SEQ ID NO:10; and

[0012] The heavy chain variable region VH2 includes HCDR1 as shown in SEQ ID NO:61, HCDR2 as shown in SEQ ID NO:46, and HCDR3 as shown in SEQ ID NO:65, and the light chain variable region VL2 includes LCDR1 as shown in SEQ ID NO:63, LCDR2 as shown in SEQ ID NO:64, and LCDR3 as shown in SEQ ID NO:50.

[0013] The drug is Eribulin, which is linked to an anti-ROR1 antibody via a linker.

[0014] In some embodiments, the heavy chain variable region VH1 contains the amino acid sequence shown in SEQ ID NO:38, the light chain variable region VL1 contains the amino acid sequence shown in SEQ ID NO:23, the heavy chain variable region VH2 contains the amino acid sequence shown in SEQ ID NO:72, and the light chain variable region VL2 contains the amino acid sequence shown in SEQ ID NO:71.

[0015] In some embodiments, the heavy chain variable region VH1 contains a sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:38, the light chain variable region VL1 contains a sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:23, the heavy chain variable region VH2 contains a sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:72, and the light chain variable region VL2 contains a sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:71.

[0016] In some embodiments, the amino acid sequence of the heavy chain variable region VH1 is shown in SEQ ID NO:38, the amino acid sequence of the light chain variable region VL1 is shown in SEQ ID NO:23, the amino acid sequence of the heavy chain variable region VH2 is shown in SEQ ID NO:72, and the amino acid sequence of the light chain variable region VL2 is shown in SEQ ID NO:71.

[0017] In some embodiments, the anti-ROR1 antibody comprises four chains as shown in (a)-(d) below:

[0018] (a) [heavy chain variable region VH1]-[CH1]-[Fc1],

[0019] (b) [Light chain variable region VL1]-[CL1],

[0020] (c)[heavy chain variable region VH2]-[CH1]-[Fc2], and

[0021] (d)[Light chain variable region VL2]-[CL2];

[0022] Alternatively, the anti-ROR1 antibody may comprise the four chains shown in (e), (b), (f), and (d) below.

[0023] (e)[heavy chain variable region VH1]-[CH1]-[Fc2],

[0024] (b) [Light chain variable region VL1]-[CL1],

[0025] (f)[heavy chain variable region VH2]-[CH1]-[Fc1], and

[0026] (d)[Light chain variable region VL2]-[CL2];

[0027] In this context, the structures shown in formulas (a), (b), (c), (d), (e), and (f) are arranged from the N-terminus to the C-terminus; CL1 and CL2 are each independently the light chain constant region of the antibody, CH1 is the first part of the heavy chain constant region of the antibody; Fc1 and Fc2 are subunits of antibody Fc, wherein Fc1 has a knotted structure according to the knife-in-hole technique and Fc2 has a hole structure according to the knife-in-hole technique, and Fc1 and Fc2 can associate with each other.

[0028] In some embodiments, the amino acid at position 366 of Fc1 is W; and the amino acid at position 366 of Fc2 is S, the amino acid at position 368 is A, and the amino acid at position 407 is V, numbered according to the EU index.

[0029] In some embodiments, Fc1 comprises an amino acid sequence as shown in SEQ ID NO: 79; and Fc2 comprises an amino acid sequence as shown in SEQ ID NO: 80.

[0030] In some embodiments, CH1 is the CH1 sequence of IgG. In some embodiments, CH1 is the CH1 of IgG1. In some embodiments, CH1 comprises the amino acid sequence shown in SEQ ID NO: 88.

[0031] In some embodiments, CL1 or CL2 is a light chain constant region of kappa or lamabda. In some embodiments, CL1 and / or CL2 comprises an amino acid sequence as shown in SEQ ID NO: 12.

[0032] In some embodiments, the anti-ROR1 antibody comprises the four chains described below:

[0033] Chain 1, comprising an amino acid sequence as shown in SEQ ID NO:81, or comprising a sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:81;

[0034] Chain 2, comprising an amino acid sequence as shown in SEQ ID NO:82, or comprising a sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:82;

[0035] Chain 3, comprising an amino acid sequence as shown in SEQ ID NO:83, or comprising a sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:83; and

[0036] Chain 4, comprising the amino acid sequence shown in SEQ ID NO:84, or comprising a sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:84; or

[0037] The anti-ROR1 antibody comprises the following four chains:

[0038] Chain 1, comprising an amino acid sequence of positions 1-446 as shown in SEQ ID NO:81, or comprising a sequence having at least 85% sequence identity with the amino acid sequence of positions 1-446 shown in SEQ ID NO:81;

[0039] Chain 2, comprising an amino acid sequence as shown in SEQ ID NO:82, or comprising a sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:82;

[0040] Chain 3, comprising an amino acid sequence of positions 1-447 as shown in SEQ ID NO:83, or comprising a sequence having at least 85% sequence identity with the amino acid sequence of positions 1-447 shown in SEQ ID NO:83; and

[0041] Chain 4, comprising an amino acid sequence as shown in SEQ ID NO:84, or comprising a sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:84.

[0042] In some embodiments, the anti-ROR1 antibody comprises the four chains described below:

[0043] Chain 1, which contains the amino acid sequence shown in SEQ ID NO:81;

[0044] Chain 2, which contains the amino acid sequence shown in SEQ ID NO:82;

[0045] Chain 3, comprising the amino acid sequence shown in SEQ ID NO:83; and

[0046] Chain 4, which contains the amino acid sequence shown in SEQ ID NO:84; or

[0047] The anti-ROR1 antibody comprises the following four chains:

[0048] Chain 1, which contains an amino acid sequence from position 1 to 446 as shown in SEQ ID NO:81;

[0049] Chain 2, which contains the amino acid sequence shown in SEQ ID NO:82;

[0050] Chain 3, comprising the amino acid sequence from position 1 to 447 as shown in SEQ ID NO:83; and

[0051] Chain 4, which contains the amino acid sequence shown in SEQ ID NO:84.

[0052] In some embodiments, the anti-ROR1 antibody comprises the four chains described below:

[0053] Chain 1, which contains a sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:81;

[0054] Chain 2, which contains a sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:82;

[0055] Chain 3, comprising a sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:83; and

[0056] Chain 4, comprising a sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:84; or

[0057] The anti-ROR1 antibody comprises the following four chains:

[0058] Chain 1, comprising a sequence having at least 85% sequence identity with the amino acid sequence from position 1 to 446 shown in SEQ ID NO:81;

[0059] Chain 2, comprising an amino acid sequence having at least 85% sequence identity as shown in SEQ ID NO:82;

[0060] Chain 3, comprising an amino acid sequence having at least 85% sequence identity from positions 1 to 447 as shown in SEQ ID NO:83; and

[0061] Chain 4, which contains a sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:84.

[0062] In some embodiments, the anti-ROR1 antibody comprises the four chains described below:

[0063] Chain 1, whose amino acid sequence is shown in SEQ ID NO:81;

[0064] Chain 2, whose amino acid sequence is shown in SEQ ID NO:82;

[0065] Chain 3, whose amino acid sequence is shown in SEQ ID NO:83; and

[0066] Chain 4, whose amino acid sequence is shown in SEQ ID NO:84.

[0067] In some embodiments, the anti-ROR1 antibody is a KIH YR8 / ZH3 antibody, the sequence of which is shown below:

[0068] Chain 1 of KIH YR8 / ZH3 (SEQ ID NO:81):

[0069] Chain 2 of KIH YR8 / ZH3 (SEQ ID NO:82):

[0070] Chain 3 of KIH YR8 / ZH3 (SEQ ID NO:83):

[0071] Chain 4 of KIHYR8 / ZH3 (SEQ ID NO:84):

[0072] In some embodiments, the anti-ROR1 antibody-drug conjugate has the following structure:

[0073] in:

[0074] Ab is an anti-ROR1 antibody; L is an enzyme-cleavable linker, and the linker forms a bond with the sulfur atom of the anti-ROR1 antibody; D is the drug eryribulin; n is an integer or decimal from 1 to 10.

[0075] In some implementations, the L has -L a -L b -L c -L d - structure, where L a As an extension unit, L b L is a spacer unit or does not exist. c If it is an oligopeptide unit or not present, L d For spacer units or L d It does not exist, and L aLinked to antibodies, L d Linked to drugs; when L d When it does not exist, L c Directly related to drugs, and so on.

[0076] In some implementations, the L has -L a -L b -L c -L d - structure, L a Linked to antibodies, L d Linked to drugs, where: L a for L b The expression is -(CH2)mC(O)-, where m is an integer from 1 to 5, and L... c L is an oligopeptide acid unit composed of 1 to 7 amino acids. d for

[0077] In some embodiments, the L has a structure selected from the group consisting of:

[0078] i)L a for L b For -C(O)- or -C(O)-NH-(CH2)2-C(O)-, L c For -glycine-glycine-phenylalanine-glycine-, -valine-citrulline-, -glycine- or bonds, and L d for -NH-CH2-O-CH2-C(O)- or bond; or

[0079] ii)L a for L b The expression is -(CH2)mC(O)-, where m is 2 or 5, and L c For -valine-citrulline- or -glycine-, and L d for or

[0080] iii)L a for L b For -NH-(CH2-CH2-O)4-(CH2)2-C(O)-, -NH-(CH2)2-C(O)- or bonds, L c For -valine-citrulline-, -glycine-, -glycine-glycine-phenylalanine-glycine- or bonds, and L d for Or -NH-CH2-O-CH2-C(O)-; and

[0081] iiii)L a for L b For -(CH2)3-C(O)-, L c For -valine-citrulline-, and L d for

[0082] Among them, wavy lines * indicates the connection point with Ab, and * indicates the connection point with L. b The connection point, a * Indicates with L c The connection point, b * Indicates the connection point with the drug.

[0083] In some embodiments, the L has the following structure:

[0084] L a for L b For -C(O)-, L c For -valine-citrulline-, and L d for

[0085] In some embodiments, the L has the following structure:

[0086] Where 1 represents the binding site with the anti-ROR1 antibody and 2 represents the binding site with the drug.

[0087] In some embodiments, the L has the following structure:

[0088] Where 1 represents the binding site with the anti-ROR1 antibody and 2 represents the binding site with the drug.

[0089] In some embodiments, the anti-ROR1 antibody-drug conjugate has the following structure:

[0090] in:

[0091] Ab is an anti-ROR1 antibody, which contains the four chains described below:

[0092] Chain 1, with the amino acid sequence shown in SEQ ID NO:81; chain 2, with the amino acid sequence shown in SEQ ID NO:82; chain 3, with the amino acid sequence shown in SEQ ID NO:83; and chain 4, with the amino acid sequence shown in SEQ ID NO:84; or comprising four chains as described below:

[0093] Chain 1, comprising an amino acid sequence of positions 1-446 as shown in SEQ ID NO:81; chain 2, comprising an amino acid sequence of positions 1-447 as shown in SEQ ID NO:83; and chain 4, comprising an amino acid sequence of positions 1-447 as shown in SEQ ID NO:84.

[0094] L is a connector, which has the following structure. Where 1 represents the binding site with the anti-ROR1 antibody, and 2 represents the binding site with the drug;

[0095] D stands for Eriblin;

[0096] n is an integer or decimal from 1 to 10.

[0097] In some embodiments, the linker-drug (LD) is selected from:

[0098] In some embodiments, the anti-ROR1 antibody-drug conjugate has the following structure:

[0099] Where n is 3 to 5.

[0100] In some implementations, n is 3.5 to 4.5.

[0101] In some embodiments, the anti-ROR1 antibody-drug conjugate is ADC-3, with the following specific structural formula:

[0102] Wherein, n is 3-5, and the anti-ROR1 antibody is a KIH YR8 / ZH3 antibody. In some embodiments, n is 3.5-4.5.

[0103] In some embodiments, the concentration of the anti-ROR1 antibody-drug conjugate is 2 mg / mL to 40 mg / mL, for example, 2 mg / mL to 30 mg / mL, 2 mg / mL to 20 mg / mL, 5 mg / mL to 40 mg / mL, 5 mg / mL to 30 mg / mL, 5 mg / mL to 20 mg / mL, or 5 mg / mL to 15 mg / mL; non-limiting examples include about 5 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, or any value between any two values. In some embodiments, the concentration of the anti-ROR1 antibody-drug conjugate is 5 mg / mL to 25 mg / mL. In some embodiments, the concentration of the anti-ROR1 antibody-drug conjugate is 5 mg / mL to 15 mg / mL. In some embodiments, the concentration of the anti-ROR1 antibody-drug conjugate is 10 mg / mL.

[0104] In some embodiments, the buffer in the pharmaceutical composition is a citrate buffer. In some embodiments, the citrate buffer is a citrate-sodium citrate buffer or a citrate-sodium hydroxide buffer. In some embodiments, the concentration of the buffer is 10 mM to 50 mM; in some embodiments, the concentration of the buffer is, for example, 10 mM to 15 mM, 15 mM to 30 mM, or 15 mM to 50 mM; non-limiting examples include about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, or any value between any two values. In some embodiments, the concentration of the buffer is 20 mM.

[0105] In some embodiments, the pH of the pharmaceutical composition is 5.0 to 7.0, for example 5.0 to 5.5, 5.0 to 6.0, 5.5 to 6.0, 5.5 to 6.5, or 6.5 to 7.0; non-limiting examples include about 5.0, about 5.5, about 6.0, about 6.5, or any value between any two values.

[0106] In some embodiments, the stabilizer of the pharmaceutical composition is sucrose, trehalose, mannitol, or a combination thereof. In some embodiments, the stabilizer is sucrose. In some embodiments, the concentration of the stabilizer is 60 mg / mL to 100 mg / mL. In some embodiments, the concentration of the stabilizer is, for example, 60 mg / mL to 70 mg / mL, 70 mg / mL to 90 mg / mL, 80 mg / mL to 100 mg / mL; non-limiting examples include about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 65 mg / mL, about 70 mg / mL, about 75 mg / mL, about 80 mg / mL, about 85 mg / mL, about 90 mg / mL, about 95 mg / mL, about 100 mg / mL, or any value between any two of these values. In some embodiments, the concentration of the stabilizer is 80 mg / mL.

[0107] In some embodiments, the stabilizer is a combination of sucrose and mannitol; wherein the concentration of sucrose is, for example, 5 mg / mL to 20 mg / mL, and non-limiting examples include about 5 mg / mL, about 10 mg / mL, 15 mg / mL, about 20 mg / mL, or any value between any two values; the concentration of mannitol is 30 to 60 mg / mL. In some embodiments, the concentration of mannitol is, for example, 30 to 40 mg / mL, 40 to 50 mg / mL, or 50 to 60 mg / mL, and non-limiting examples include about 30 mg / mL, about 40 mg / mL, about 45 mg / mL, about 50 mg / mL, about 60 mg / mL, or any value between any two values. In some embodiments, the stabilizer is a combination of 15 mg / mL sucrose and 45 mg / mL mannitol.

[0108] In some embodiments, the stabilizer is a combination of trehalose and mannitol; wherein the concentration of trehalose is, for example, 5 mg / mL to 20 mg / mL, and non-limiting examples include about 5 mg / mL, about 10 mg / mL, 15 mg / mL, about 20 mg / mL, or any value between any two values; the concentration of mannitol is 30 to 60 mg / mL. In some embodiments, the concentration of mannitol is, for example, 30 to 40 mg / mL, 40 to 50 mg / mL, or 50 to 60 mg / mL, and non-limiting examples include about 30 mg / mL, about 40 mg / mL, about 45 mg / mL, about 50 mg / mL, about 60 mg / mL, or any value between any two values. In some embodiments, the stabilizer is a combination of 15 mg / mL trehalose and 45 mg / mL mannitol.

[0109] In some embodiments, the surfactant in the pharmaceutical composition is polysorbate. In some embodiments, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the concentration of the surfactant is 0.01 mg / mL to 1.0 mg / mL. In some embodiments, the concentration of the buffer is, for example, 0.01 mg / mL to 0.1 mg / mL, 0.1 mg / mL to 0.5 mg / mL, or 0.5 mg / mL to 1 mg / mL; non-limiting examples include about 0.02 mg / mL, about 0.05 mg / mL, about 0.1 mg / mL, about 0.15 mg / mL, about 0.2 mg / mL, about 0.25 mg / mL, about 0.3 mg / mL, about 0.35 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, about 1.0 mg / mL, or any value between any two of these values. In some embodiments, the concentration of the buffer is 0.2 mg / mL.

[0110] In some embodiments, the pharmaceutical composition comprises:

[0111] a) Anti-ROR1 antibody-drug conjugate at concentrations of 2 mg / mL to 40 mg / mL;

[0112] b) 10mM~50mM citrate buffer;

[0113] c) Stabilizer at concentrations of 5 mg / mL to 100 mg / mL;

[0114] d) 0.1 mg / mL to 0.5 mg / mL surfactant; the pH value of the pharmaceutical composition is 5.0 to 7.0.

[0115] In some embodiments, the pharmaceutical composition comprises:

[0116] a) Anti-ROR1 antibody-drug conjugate at concentrations of 2 mg / mL to 40 mg / mL;

[0117] b) 10mM~50mM citrate buffer;

[0118] c) 60 mg / mL to 100 mg / mL sucrose or trehalose;

[0119] d) 0.1 mg / mL to 0.5 mg / mL surfactant; the pH value of the pharmaceutical composition is 5.0 to 7.0;

[0120] or

[0121] The pharmaceutical composition comprises:

[0122] a) Anti-ROR1 antibody-drug conjugate at concentrations of 2 mg / mL to 40 mg / mL;

[0123] b) 10mM~50mM citrate buffer;

[0124] c) 5 mg / mL to 20 mg / mL sucrose and 30 mg / mL to 60 mg / mL mannitol;

[0125] d) 0.1 mg / mL to 0.5 mg / mL surfactant; the pH value of the pharmaceutical composition is 5.0 to 7.0.

[0126] In some embodiments, the pharmaceutical composition comprises:

[0127] a) Anti-ROR1 antibody-drug conjugate at concentrations of 2 mg / mL to 40 mg / mL;

[0128] b) 10mM~50mM citrate buffer;

[0129] c) Stabilizer at 70 mg / mL to 90 mg / mL;

[0130] d) 0.1 mg / mL to 0.5 mg / mL surfactant; the pH value of the pharmaceutical composition is 5.0 to 7.0.

[0131] In some embodiments, the pharmaceutical composition comprises:

[0132] a) 5 mg / mL to 15 mg / mL anti-ROR1 antibody-drug conjugate;

[0133] b) 15mM~30mM citrate buffer;

[0134] c) 70 mg / mL–90 mM mg / mL sucrose or trehalose; and

[0135] d) 0.1 mg / mL to 0.5 mg / mL polysorbate 20 or polysorbate 80, wherein the pH of the pharmaceutical composition is 5.0 to 6.5.

[0136] In some embodiments, the pharmaceutical composition comprises:

[0137] a) 5 mg / mL to 15 mg / mL anti-ROR1 antibody-drug conjugate;

[0138] b) 15mM~30mM citrate buffer;

[0139] c) 5 mg / mL–20 mg / mL sucrose and 30 mg / mL–60 mg / mL mannitol; and

[0140] d) 0.1 mg / mL to 0.5 mg / mL polysorbate 20 or polysorbate 80, wherein the pH of the pharmaceutical composition is 5.0 to 6.5.

[0141] In some embodiments, the pharmaceutical composition comprises:

[0142] a) 10 mg / mL anti-ROR1 antibody-drug conjugate;

[0143] b) 20mM citrate-sodium citrate buffer;

[0144] c) 80 mg / mL sucrose; and

[0145] d) 0.2 mg / mL polysorbate 20, wherein the pH of the pharmaceutical composition is 5.5–6.5; or

[0146] The pharmaceutical composition comprises:

[0147] a) 10 mg / mL anti-ROR1 antibody-drug conjugate;

[0148] b) 20mM citrate-sodium citrate buffer;

[0149] c) 15 mg / mL sucrose and 45 mg / mL mannitol; and

[0150] d) 0.2 mg / mL polysorbate 20, wherein the pH of the pharmaceutical composition is 5.5 to 6.5.

[0151] In some embodiments, the pharmaceutical composition comprises:

[0152] a) 10 mg / mL anti-ROR1 antibody-drug conjugate;

[0153] b) 20mM citrate-sodium citrate buffer;

[0154] c) 80 mg / mL sucrose; and

[0155] d) 0.2 mg / mL polysorbate 20, wherein the pH of the pharmaceutical composition is 5.0–6.5; or

[0156] The pharmaceutical composition comprises:

[0157] a) 10 mg / mL anti-ROR1 antibody-drug conjugate;

[0158] b) 20mM citrate-sodium citrate buffer;

[0159] c) 15 mg / mL sucrose and 45 mg / mL mannitol; and

[0160] d) 0.2 mg / mL polysorbate 20, wherein the pH of the pharmaceutical composition is 5.0 to 6.5.

[0161] In some embodiments, the pharmaceutical composition comprises the following components:

[0162] a) 10 mg / mL anti-ROR1 antibody-drug conjugate;

[0163] b) 20mM citrate-sodium citrate buffer;

[0164] c) 80 mg / mL sucrose; and

[0165] d) 0.2 mg / mL polysorbate 20, wherein the pH of the pharmaceutical composition is 5.5 to 6.5.

[0166] In some embodiments, the pharmaceutical composition comprises the following components:

[0167] a) 10 mg / mL anti-ROR1 antibody-drug conjugate;

[0168] b) 20mM citrate-sodium citrate buffer;

[0169] c) 15 mg / mL sucrose and 45 mg / mL mannitol; and

[0170] d) 0.2 mg / mL polysorbate 20, wherein the pH of the pharmaceutical composition is 5.5 to 6.5.

[0171] In some embodiments, the pharmaceutical composition comprises the following components:

[0172] a) 10 mg / mL anti-ROR1 antibody-drug conjugate;

[0173] b) 20mM citrate-sodium citrate buffer;

[0174] c) 80 mg / mL sucrose; and

[0175] d) 0.2 mg / mL polysorbate 20, wherein the pH of the pharmaceutical composition is 5.0 to 6.0.

[0176] In some embodiments, the pharmaceutical composition comprises the following components:

[0177] a) 10 mg / mL anti-ROR1 antibody-drug conjugate;

[0178] b) 20mM citrate-sodium citrate buffer;

[0179] c) 15 mg / mL sucrose and 45 mg / mL mannitol; and

[0180] d) 0.2 mg / mL polysorbate 20, wherein the pH of the pharmaceutical composition is 5.0 to 6.0.

[0181] In some embodiments, the pharmaceutical composition comprises:

[0182] a) 10 mg / mL anti-ROR1 antibody-drug conjugate;

[0183] b) 20mM citrate-sodium citrate buffer;

[0184] c) 80 mg / mL sucrose; and

[0185] d) 0.2 mg / mL polysorbate 20, wherein the pH of the pharmaceutical composition is 5.5.

[0186] In some embodiments, the pharmaceutical composition comprises the following components:

[0187] a) 10 mg / mL anti-ROR1 antibody-drug conjugate;

[0188] b) 20mM citrate-sodium citrate buffer;

[0189] c) 80 mg / mL sucrose; and

[0190] d) 0.2 mg / mL polysorbate 20, wherein the pH of the pharmaceutical composition is 5.5.

[0191] It should be understood that the pharmaceutical compositions protected by this disclosure are not limited to the combinations described above. Any combination of technical solutions that can accomplish the purposes of this disclosure is within the scope of protection of this disclosure.

[0192] In some embodiments, the pharmaceutical composition is a liquid formulation.

[0193] In a second aspect, a method for preparing the pharmaceutical composition disclosed herein is provided, comprising accurately weighing various components according to the formulation of the composition, preparing it with water for injection, and adjusting the pH value. Optionally, the method includes a step of replacing the stock solution of the anti-ROR1 antibody-drug conjugate composition with a buffer, wherein the buffer is preferably a citrate buffer or a citrate-sodium citrate buffer.

[0194] The third aspect of this disclosure discloses a lyophilized formulation prepared by freeze-drying the above-described pharmaceutical composition.

[0195] In some embodiments, the freeze-drying includes the steps of pre-freezing, primary drying, and secondary drying. In some embodiments, the pre-freezing step includes the following steps:

[0196] a1. Cool the above-mentioned pharmaceutical composition to -40°C or lower;

[0197] a2. Maintain isothermal temperature for at least 120 min; and / or

[0198] The first drying process includes the following steps:

[0199] b1. Warm the pharmaceutical composition from -40°C or lower to -20°C;

[0200] b2. Maintain isothermal temperature for at least 25 hours.

[0201] In some implementations, the primary drying process includes the following steps:

[0202] b1. Warm the pharmaceutical composition from -40°C or lower to -20°C;

[0203] b2. Isothermal holding for 25-50 hours, 30-50 hours or 40-50 hours, including but not limited to isothermal holding for 30 hours, 35 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 ​​hours, 49 hours or 50 hours.

[0204] In some implementations, the primary drying process includes the following steps:

[0205] b1. Warm the pharmaceutical composition from -40°C or lower to -20°C;

[0206] b2. Maintain isothermal temperature for 40-50 hours.

[0207] In some implementations, the pre-freezing step does not include the annealing step; specifically, the pre-freezing step is as follows:

[0208] a1. Cool the above-mentioned pharmaceutical composition to -40°C;

[0209] a2. Maintain isothermal temperature for 120 minutes.

[0210] In some implementations, the pre-freezing step further includes an annealing step:

[0211] a3. Anneal at a temperature of -40°C or lower to -10°C;

[0212] a4. Maintain isothermal temperature for at least 120 minutes;

[0213] a5. Adjust the temperature from -10℃ to -40℃ or lower;

[0214] a6. Maintain isothermal temperature for at least 120 minutes.

[0215] In some implementations, the pre-freezing step further includes an annealing step:

[0216] a3. Anneal at a temperature of -40°C to -10°C;

[0217] a4. Maintain isothermal temperature for at least 60 minutes;

[0218] a5. Adjust from -10℃ to -40℃;

[0219] a6. Maintain isothermal temperature for at least 120 minutes.

[0220] In some implementations, secondary drying includes the following steps:

[0221] c1. Increase the temperature to 25-35℃;

[0222] c2. Maintain isothermal temperature for at least 600 minutes.

[0223] It should be understood that other additional steps may be included before step a1, between steps a1 and a2, between steps a2 and a3, between steps a3 and a4, between steps a4 and a5, between steps a5 and a6, between steps a6 and b1, between steps b1 and b2, between steps b2 and c1, between steps c1 and c2, or after step c2 to complete the technical solution disclosed herein, and all of these are within the scope of protection of this disclosure.

[0224] In some embodiments, the cooling rate in step a1 is 0.1°C / min to 1.0°C / min. In some embodiments, the cooling rate is, for example, 0.1°C / min to 0.2°C / min, 0.2°C / min to 0.5°C / min, or 0.5°C / min to 1.0°C / min; non-limiting embodiments include about 0.2°C / min, about 0.4°C / min, about 0.45°C / min, about 0.5°C / min, about 0.6°C / min, about 0.8°C / min, about 1.0°C / min, or any value between any two of these values. In some embodiments, the cooling rate is 0.45°C / min.

[0225] In some embodiments, the heating rate in step a3 is 0.1°C / min to 1.0°C / min. In other embodiments, the heating rate is, for example, 0.1°C / min to 0.2°C / min, 0.2°C / min to 0.5°C / min, or 0.5°C / min to 1.0°C / min. Non-limiting embodiments include about 0.2°C / min, about 0.4°C / min, about 0.45°C / min, about 0.5°C / min, about 0.6°C / min, about 0.8°C / min, about 1.0°C / min, or any value between any two of these values. In some embodiments, the heating rate is 0.5°C / min.

[0226] In some embodiments, the cooling rate in step a5 is 0.1°C / min to 1.0°C / min. In other embodiments, the cooling rate is, for example, 0.1°C / min to 0.2°C / min, 0.2°C / min to 0.5°C / min, or 0.5°C / min to 1.0°C / min. Non-limiting embodiments include about 0.2°C / min, about 0.4°C / min, about 0.45°C / min, about 0.5°C / min, about 0.6°C / min, about 0.8°C / min, about 1.0°C / min, or any value between any two of these values. In some embodiments, the cooling rate is 0.5°C / min.

[0227] In some embodiments, the pharmaceutical composition described above is cooled to -40°C in step a1.

[0228] In some implementations, isothermal holding in step a2 is 120-250 min, preferably 120 min.

[0229] In some implementations, isothermal holding in step a4 is 120-250 min, preferably 120 min.

[0230] In some implementations, the temperature is lowered to -40°C in step a5.

[0231] In some implementations, step a6 involves isothermal holding for 120-250 minutes, preferably 120 minutes.

[0232] In some implementations, step b2 involves isothermal holding for 2820 min (i.e., 47 hours).

[0233] In some embodiments, the vacuum level of the primary and secondary drying processes is less than 0.12 mbar; non-limiting embodiments include about 0.04 mbar, about 0.06 mbar, about 0.08 mbar, about 0.10 mbar, and about 0.12 mbar, and in some specific embodiments, the vacuum level of the primary and secondary drying processes is 0.08 mbar.

[0234] In some embodiments, a pre-cooling step is included before the pre-freezing step. The pre-cooling step includes pre-cooling the shelf to 5°C and maintaining it at 5°C for 50-80 minutes. In some embodiments, it is maintained at 5°C for 60 minutes.

[0235] The fourth aspect of this disclosure discloses a reconstituted solution, which is prepared by reconstituted the above-mentioned lyophilized formulation.

[0236] In some embodiments, the reconstitution solution comprises:

[0237] a) 5 mg / mL to 15 mg / mL anti-ROR1 antibody-drug conjugate;

[0238] b) 15mM~30mM citrate buffer;

[0239] c) 70 mg / mL to 90 mg / mL sucrose or trehalose; and

[0240] d) 0.1 mg / mL to 0.5 mg / mL polysorbate 20 or polysorbate 80, wherein the pH of the pharmaceutical composition is 5.0 to 6.5; or

[0241] a) 5 mg / mL to 15 mg / mL anti-ROR1 antibody-drug conjugate;

[0242] b) 15mM~30mM citrate buffer;

[0243] c) 5 mg / mL–20 mg / mL sucrose and 30 mg / mL–60 mg / mL mannitol; and

[0244] d) 0.1 mg / mL to 0.5 mg / mL polysorbate 20 or polysorbate 80, wherein the pH of the pharmaceutical composition is 5.0 to 6.5.

[0245] In some embodiments, the reconstitution solution comprises:

[0246] a) 10 mg / mL anti-ROR1 antibody-drug conjugate;

[0247] b) 20mM citric acid-sodium citrate;

[0248] c) 80 mg / mL sucrose; and

[0249] d) 0.2 mg / mL polysorbate 20, wherein the pH of the pharmaceutical composition is 5.0–6.5; or

[0250] a) 10 mg / mL anti-ROR1 antibody-drug conjugate;

[0251] b) 20mM citrate-sodium citrate buffer;

[0252] c) 15 mg / ml sucrose and 45 mg / ml mannitol; and

[0253] d) 0.2 mg / mL polysorbate 20, wherein the pH of the pharmaceutical composition is 5.0 to 6.5.

[0254] In some embodiments, the reconstitution solution comprises:

[0255] a) 10 mg / mL anti-ROR1 antibody-drug conjugate;

[0256] b) 20mM citrate-sodium citrate buffer;

[0257] c) 80 mg / mL sucrose; and

[0258] d) 0.2 mg / mL polysorbate 20, wherein the pH of the pharmaceutical composition is 5.5.

[0259] The fifth aspect of this disclosure discloses a method for preparing a reconstituted solution containing an anti-ROR1 antibody-drug conjugate, which includes reconstituted a lyophilized formulation to obtain the reconstituted solution.

[0260] In some implementations, water for injection is used to reconstitute the lyophilized formulation.

[0261] In a sixth aspect, an article is provided comprising a container containing a pharmaceutical composition, lyophilized preparation, or reconstituted solution as described above.

[0262] When intended for in vivo administration, the formulations disclosed herein shall be sterile. The formulations disclosed herein may be sterilized by various sterilization methods, including sterile filtration, radiation, etc. In one embodiment, the antibody formulation is sterilized by filtration through a pre-sterilized 0.22-micron filter. The sterile composition for injection may be formulated according to standard pharmaceutical practice, as described in Remington: The Science & Practice of Pharmacy, 21st edition, Lippincott Williams & Wilkins, (2005). Formulations containing antibody-drug conjugates (such as those disclosed herein) may generally be stored in lyophilized form or in solution. Sterile compositions containing antibody-drug conjugates are intended to be placed in containers with sterile inlets, such as intravenous solution packs or vials with connectors that allow retrieval of the formulation, such as stoppers pierced by a hypodermic needle. In one embodiment, the pharmaceutical composition disclosed herein is provided in the form of a pre-filled syringe.

[0263] In a seventh aspect, the use of the pharmaceutical compositions, lyophilized formulations, or reconstituted solutions disclosed herein in the preparation of medicaments for treating diseases is provided. In some embodiments, the disease is a tumor or cancer.

[0264] In an eighth aspect, the pharmaceutical compositions, lyophilized formulations, or reconstituted solutions disclosed herein are provided for use as medicaments for treating diseases. In some embodiments, the disease is a tumor or cancer.

[0265] In a ninth aspect, this disclosure provides a method of treating a disease, comprising administering to a subject in need an effective amount of the pharmaceutical composition of this disclosure, the lyophilized formulation of this disclosure, or the reconstituted solution of this disclosure. In some embodiments, the disease is a tumor or cancer.

[0266] In some embodiments, the tumor or cancer is a solid tumor or a hematologic malignancy. In some embodiments, the tumor or cancer is selected from breast cancer, pancreatic cancer, lung cancer, esophageal cancer, laryngeal tumors, sarcomas, pharyngeal tumors, oral tumors, gastric cancer, ovarian cancer, prostate cancer, bladder cancer, colon cancer, colorectal cancer, urothelial carcinoma, neuroblastoma, cervical cancer, lymphoma, and leukemia.

[0267] In some implementations, the tumor or cancer is a hematologic malignancy, including lymphoma and leukemia.

[0268] In some implementations, the lymphomas and leukemias include non-Hodgkin lymphoma, Hodgkin lymphoma, acute lymphoblastic leukemia, anaplastic large cell lymphoma, B-cell chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), myeloid hematologic leukemia, pre-B-cell acute lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), and marginal zone lymphoma (MZL).

[0269] In some implementations, the lung cancer is non-small cell lung cancer.

[0270] In some implementations, the tumor or cancer expresses ROR1.

[0271] The pharmaceutical composition disclosed herein exhibits excellent physical and chemical stability, with outstanding stability under conditions such as high temperature and light exposure, and can be stored stably for a long period of time. Attached Figure Description

[0272] Figure 1 shows the tumor-suppressive activity of ADC in the HCC1187 breast cancer model of female SCID mice.

[0273] Figure 2 shows the tumor suppressor activity of ADC in the Jeko-1 mantle cell lymphoma model of female SCID mice.

[0274] Figure 3 shows the tumor-suppressive activity of ADC in a mouse model transplanted with the PA-1 ovarian cancer cell line. Detailed Implementation

[0275] The term “comprising” encompasses both the meaning of “including” and “consisting of”. For example, a composition that “comprising” X may consist of only X or may include other components, such as X+Y.

[0276] The singular forms “a” and “an” used in the specification and claims include the corresponding plural references, unless otherwise clearly stated in the content.

[0277] “Composition” means a mixture containing one or more of the anti-ROR1 antibody-drug conjugates disclosed herein, along with other chemical components, such as physiological / pharmaceutical-grade carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertment of its biological activity. In this disclosure, “pharmaceutical composition,” “composition,” “formulation,” and “prescription” are used interchangeably. Unless otherwise specified, the solvent in the solution form of the pharmaceutical compositions disclosed herein is water.

[0278] "Buffer" refers to a substance that is resistant to pH changes through the action of its acid-base conjugate components. Suitable buffering agents for use in the compositions disclosed herein include, but are not limited to, citrate-sodium citrate buffers or citrate buffers.

[0279] "Stabilizer" refers to a substance that can increase the stability of a pharmaceutical composition. Stabilizers help prevent the oxidation and aggregation of proteins in pharmaceutical compositions, especially liquid pharmaceutical compositions. Typical stabilizers used in this disclosure include, but are not limited to, sugars, including, but not limited to, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbitol, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as raffinose, mesotriose, maltodextrin, dextran, starch, etc.; and sugar alcohols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucosol), etc. Sugars can be sugar alcohols or amino sugars. Preferably, the sugar is not a reducing sugar. Reducing sugars include, but are not limited to, all monosaccharides, lactose, maltose, and cellobiose. Therefore, the sugar is preferably a non-reducing sugar, such as sucrose, trehalose, raffinose, sorbitol, and mannitol, more preferably sucrose.

[0280] Surfactants suitable for the compositions disclosed herein include, but are not limited to, nonionic surfactants, ionic surfactants, amphoteric surfactants, and combinations thereof. Typical surfactants used in this disclosure include, but are not limited to, sorbitan fatty acid esters (e.g., sorbitan monocaprylate, sorbitan monolaurate, sorbitan monopalmitate), sorbitan trioleate, glycerol fatty acid esters (e.g., glycerol monocaprylate, glycerol monomyristate, glycerol monostearate), polyglycerol fatty acid esters (e.g., decaglyceryl monostearate, decaglyceryl distearate, decaglyceryl monolinoleate), and polyoxyethylene sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monostea ... Polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, polyoxyethylene sorbitan tristearate, polyoxyethylene sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan tetrastearate, polyoxyethylene sorbitan tetraoleate), polyoxyethylene glycerol fatty acid esters (e.g., polyoxyethylene glycerol monostearate), polyethylene glycol fatty acid esters (e.g., polyethylene glycol distearate), polyoxyethylene alkyl ethers (e.g., polyoxyethylene alkyl ethers). Polyoxyethylene lauryl ether, polyoxyethylene polyoxypropylene alkyl ether (e.g., polyoxyethylene polyoxypropylene glycol, polyoxyethylene polyoxypropylene propyl ether, polyoxyethylene polyoxypropylene cetyl ether), polyoxyethylene alkylphenyl ether (e.g., polyoxyethylene nonylphenyl ether), polyoxyethylene hydrogenated castor oil (e.g., polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil), polyoxyethylene beeswax derivatives (e.g., polyoxyethylene sorbitan beeswax), polyoxyethylene lanolin derivatives (e.g., polyoxyethylene lanolin), and polyoxyethylene fatty acid amides (e.g., polyoxyethylene... The composition may contain: stearamide; C10-C18 alkyl sulfates (e.g., sodium cetyl sulfate, sodium lauryl sulfate, sodium oleyl sulfate), polyoxyethylene C10-C18 alkyl ether sulfates (e.g., polyoxyethylene sodium lauryl sulfate) having an average addition of 2 to 4 moles of ethylene oxide units, and C1-C18 alkyl sulfosuccinates (e.g., sodium lauryl sulfosuccinate); and natural surfactants such as lecithin, glycerophospholipids, sphingomyelin (e.g., sphingomyelin), and sucrose esters of C12-C18 fatty acids. The composition may contain one or more of these surfactants. Preferred surfactants are polyoxyethylene sorbitan fatty acid esters, such as polysorbate 20, 40, 60, or 80. Polysorbate 20 or 80 (e.g., at a concentration of about 0.2 mg / mL) is particularly suitable.

[0281] As used herein, the terms “about” or “approximately” mean a numerical value within an acceptable margin of error for a specific value determined by a person skilled in the art, the numerical value depending in part on how it is measured or determined (i.e., the limits of the measurement system). For example, in every practice in the art, “about” may mean within or greater than 1 standard deviation. Alternatively, “about” or “substantially comprises” may mean a range of up to ±20%, for example, about 5.5 pH means pH 5.5 ± 1.1. Furthermore, particularly for biological systems or processes, the term may mean up to an order of magnitude or up to 5 times the numerical value. Unless otherwise stated, when a specific value appears in this application and claims, the meaning of “about” or “substantially comprises” should be assumed to be within an acceptable margin of error for that specific value.

[0282] The term "antibody" is used in the broadest sense and encompasses a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, full-length antibodies, and antigen-binding fragments, as long as they exhibit the desired antigen-binding activity. Typically, natural IgG antibodies are heterotetrameric proteins composed of two light chains and two heavy chains linked by disulfide bonds. From the N to C terminus, each heavy chain has one variable region (VH) and three constant domains (CH1, CH2, and CH3). From the N to C terminus, each light chain has one variable region (VL) and one constant light domain (CL). The specific meaning of "antibody" can be determined by a person skilled in the art based on the context.

[0283] An "antibody fragment" or "antigen-binding fragment" is different from a complete antibody molecule, but contains a portion of the complete antibody that retains the antigen-binding ability of the complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, single-domain antibodies, single-chain Fab (scFab), biantibodies, linear antibodies, single-chain antibodies (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0284] The complementarity-determining region (CDR) or CDR is a region within the variable domain of an antibody that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). The CDR is primarily responsible for binding to antigen epitopes. CDRs within the variable domain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus. In a given variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any of a number of known antibody CDR assignment systems or combinations thereof, including, for example: Chothia (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)) based on antibody three-dimensional structure and CDR loop topology; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, Department of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability; AbM (University of Bath); Contact (University College London); and the International ImMunoGeneTics. The database (IMGT) (http: / / imgt.cines.fr / ) and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. The correspondences between various numbering systems are well known to those skilled in the art. For example, CDRs can be obtained using the AbYsis database (www.bioinf.org.uk / abysis / sequence_input / key_annotation / key_annotation.cg). In other words, when a CDR sequence and its location in an antibody are provided under one numbering system, those skilled in the art are able to determine the corresponding CDR sequence and its location in an antibody under another numbering system. Technical solutions corresponding to different numbering systems are considered equivalent. In one embodiment, the CDR of the antibody disclosed herein is located according to the Kabat numbering scheme.

[0285] The term "bispecific antibody" refers to an antibody (including the antibody or its antigen-binding fragment, such as a single-chain antibody) that can specifically bind to two different antigens or at least two different antigenic epitopes of the same antigen. Bispecific antibodies with various structures have been disclosed in the prior art. Based on the integrity of the IgG molecule, they can be classified into IgG-like bispecific antibodies and antibody fragment-type bispecific antibodies; based on the number of antigen-binding regions, they can be classified into bivalent, trivalent, tetravalent, or more bispecific antibodies; and based on whether the structure is symmetrical, they can be classified into symmetrical and asymmetrical bispecific antibodies. Among them, bispecific antibodies based on antibody fragments, such as Fab fragments lacking the Fc fragment, form bispecific antibodies by combining two or more Fab fragments into one molecule. These antibodies have low immunogenicity, small molecular weight, and high tumor tissue penetration. Typical antibody structures of this type include F(ab)2, scFv-Fab, and (scFv)2-Fab. IgG-like bispecific antibodies (e.g., those with an Fc fragment) have a relatively large molecular weight. The Fc fragment helps in antibody purification and improves its solubility and stability. The Fc portion may also bind to the receptor FcRn, increasing the antibody's serum half-life. Typical bispecific antibody structural models include KiH, CrossMAb, Triomab quadroma, FcΔAdp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1 TCBs, and 1Fab-IgG. TDB, FynomAb, two-in-one / DAF, scFv-Fab-IgG, DART-Fc, LP-DART, CODV-Fab-TL, HLE-BiTE, F(ab)2-CrossMAb , IgG-(scFv)2, Bs4Ab, DVD-Ig, Tetravalent-DART-Fc, (scFv)4-Fc, CODV-Ig, mAb2, F(ab)4-CrossMAb, etc. (see Aran F. Labrijn et al., Nature Reviews Drug Discovery volume 18, pages 585–608 (2019); Chen S1 et al., J Immunol Res. 2019 Feb 11; 2019:4516041).

[0286] The term "antibody-drug conjugate" (ADC) refers to an antibody or antibody fragment linked to a biologically active toxic drug via a linker. The antibodies or antibody fragments described in this disclosure can be conjugated to effector molecules in any manner. For example, antibodies or antibody fragments can be attached to toxic drugs chemically or recombinantly. Chemical methods for preparing fusions or conjugates are known in the art. Methods for conjugating antibodies or antibody fragments to drugs must be able to link the antibody to the toxic drug without interfering with the ability of the antibody or antibody fragment to bind to the target molecule.

[0287] The drugs in the antibody-drug conjugates disclosed herein are cytotoxic drugs or cytotoxic compounds, which are substances that inhibit or prevent cell function and / or cause cell death or destruction. In principle, cytotoxic drugs can kill tumor cells at sufficiently high concentrations; however, due to their lack of specificity, they can also cause apoptosis of normal cells while killing tumor cells, leading to serious side effects. Cytotoxic drugs include toxins, such as small molecule toxins or enzyme-active toxins derived from bacteria, fungi, plants, or animals; radioactive isotopes (e.g., radioactive isotopes of At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, and Lu); chemotherapeutic agents; antibiotics; and ribolysins.

[0288] The antibody and the cytotoxic drug disclosed herein can be conjugated via a conjugating agent. The conjugating agent can be any one or more of the following: non-selective conjugating agents, carboxyl-based conjugating agents, peptide chains, and disulfide-based conjugating agents. The non-selective conjugating agent refers to a compound that covalently links the effector molecule and the antibody, such as glutaraldehyde. The carboxyl-based conjugating agent can be any one or more of the following: maleic aconitine-based conjugating agents (e.g., maleic aconitine) and acylhydrazone-based conjugating agents (with an acylhydrazone as the conjugation site).

[0289] The DAR value (Drug-Antibody Ratio) refers to the ratio of drug to payload molecules conjugated to each antibody. This value is usually an average value, and for example, the DAR value can be 3-6, such as 3-5, 3.5-4.5, or 4-6; or it can be 1, 2, 3, 4, 5, 6, 7, or 8, such as 3, 4, or 5; more preferably, for example, 4.

[0290] The pharmaceutical compositions disclosed herein achieve a stable effect: the antibody-drug conjugate therein substantially retains its physical and / or chemical stability and / or biological activity after storage; for example, the pharmaceutical composition substantially retains its physical and chemical stability and its biological activity after storage. The storage period is generally selected based on the intended shelf life of the pharmaceutical composition. Currently, various analytical techniques are available for measuring protein stability, which can measure stability after storage at a selected temperature for a selected period of time.

[0291] Stable antibody-drug conjugate formulations are those in which no significant changes are observed when stored at refrigerated temperatures (2-8°C) for at least 3 months, at least 6 months, at least 1 year, and up to 2 years. Additionally, stable liquid formulations include those that exhibit the desired characteristics after storage at 25°C for 1 month, 3 months, or 6 months, or at 40°C for 1 month. Typical acceptable criteria for stability are as follows: Degradation of antibody monomers typically not exceeding about 10%, e.g., not exceeding about 5%, as determined by SEC-HPLC. Visually, the antibody-drug conjugate formulation is colorless or clear to slightly milky white. The concentration, pH, and osmotic pressure of the formulation exhibit variations not exceeding ±10%. Truncation typically not exceeding about 10%, e.g., not exceeding about 5%, and aggregation typically not exceeding about 10%, e.g., not exceeding about 5%, are observed.

[0292] If, after visual inspection of color and / or clarity, or by means of UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS), the antibody-drug conjugate does not show significant increase in aggregation, precipitation, and / or denaturation, then the antibody-drug conjugate "retains its physical stability" in the pharmaceutical formulation. Changes in protein conformation can be evaluated by fluorescence spectroscopy (which determines the protein's tertiary structure) and by FTIR spectroscopy (which determines the protein's secondary structure).

[0293] If an antibody-drug conjugate does not exhibit significant chemical changes, then the antibody-drug conjugate "retains its chemical stability" in the pharmaceutical formulation. Chemical stability can be assessed by detecting and quantifying the chemically altered form of the protein. Degradation processes that frequently alter the chemical structure of proteins include hydrolysis or truncation (evaluated by methods such as size exclusion chromatography and SDS-PAGE), oxidation (evaluated by methods such as peptide mapping combined with mass spectrometry or MALDI / TOF / MS), deamidation (evaluated by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, and isofpartate measurement), and isomerization (evaluated by measuring isofpartate content, peptide mapping, etc.).

[0294] If the biological activity of an antibody-drug conjugate at a given time is within a predetermined range of the biological activity exhibited when the drug formulation is prepared, then the antibody-drug conjugate "retains its biological activity" in the drug formulation. The biological activity of an antibody-drug conjugate can be determined, for example, by an antigen binding assay.

[0295] "Displacement" refers to the replacement of the solvent system in which the antibody-drug conjugate is dissolved. For example, a high-salt or hypertonic solvent system containing the antibody-drug conjugate may be replaced by a physical manipulation of the buffer system of the stabilizing formulation, thereby allowing the antibody-drug conjugate to remain in the stabilizing formulation. The physical manipulation methods include, but are not limited to, ultrafiltration, dialysis, or reconstitution after centrifugation.

[0296] "Liquid composition" refers to an aqueous composition that is not reconstituted from a lyophilized product and contains at least one anti-ROR1 antibody-drug conjugate and at least one other excipient (e.g., buffer). This liquid composition may contain other excipients (stabilizers, surfactants) and other active ingredients. Such formulations are also known as "ready-to-use" formulations.

[0297] "Lyophilized formulation" refers to a substantially anhydrous, dried (e.g., freeze-dried) pharmaceutical composition. Antibody lyophilization technology is well-known in the art; see, for example, Rey and May (2004), Freeze-Drying / Lyophilization of Pharmaceutical & Biological Products, ISBN 0824748689. Reconstitute the lyophilized material to obtain an aqueous composition (reconstituted solution)—typically for immediate use (e.g., within 1–10 days)—because lyophilized materials often have a limited shelf life after reconstitution.

[0298] Freeze-drying typically involves a series of steps: pre-freezing, primary drying, and secondary drying. Freeze-drying is performed by freezing the formulation and subsequently sublimating water at a temperature suitable for primary drying. Under these conditions, the product temperature is below the eutectic point or collapse temperature of the formulation. Typically, the primary drying temperature ranges from about -50°C to 0°C (assuming the product remains frozen during the primary drying process). The size and type of the formulation, the container holding the sample (e.g., a glass vial), and the volume of liquid determine the required drying time, which can range from several hours to several days (e.g., 40-60 hours). The secondary drying stage can be performed, for example, from about -10°C to 40°C, depending primarily on the type and size of the container and the type of protein used. The secondary drying time is determined by the desired residual moisture level in the product and typically requires at least about 5 hours. Generally, the water content of the low-pressure freeze-dried formulation is less than about 5%, preferably less than about 3%. The pressure can be the same as the pressure applied in the primary drying step. Freeze-drying conditions can vary depending on the formulation and vial size.

[0299] When applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, "giving" and "treatment" refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Giving" and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and cells, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cells. "Giving" and "treatment" also mean the treatment of, for example, cells, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo. When applied to humans, veterinary, or research subjects, "treatment" refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

[0300] "Treatment" means administering an oral or topical therapeutic agent, such as one comprising any of the compositions disclosed herein, to a subject who has symptoms of one or more diseases, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, a therapeutic agent is administered in a treated subject or population in an amount that effectively relieves symptoms of one or more diseases to induce regression of such symptoms or inhibit their progression to any clinically measurable extent. The amount of a therapeutic agent that effectively relieves any specific disease symptom (also referred to as a "therapeuticly effective amount") can vary depending on a variety of factors, such as the subject's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the subject. Whether the disease symptoms have been relieved can be evaluated using any clinical test method commonly used by a physician or other healthcare professional to assess the severity or progression of the symptoms. Although the embodiments disclosed herein (e.g., treatment methods or products) may be ineffective in alleviating symptoms of each target disease, they should reduce symptoms of the target disease in a statistically significant number of subjects, as determined by any statistical test known in the art, such as the Student t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

[0301] An "effective dose" includes an amount sufficient to improve or prevent the symptoms or condition of a medically diagnosed disease. An effective dose also means an amount sufficient to allow or facilitate diagnosis. The effective dose for a specific subject or veterinary subject can vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. An effective dose can be the maximum dose or administration regimen that avoids significant side effects or toxicity.

[0302] "Optional" or "optionally" means that the event or circumstances described below may, but do not have to, occur, and the description includes the circumstances in which the event or circumstances may or may not occur.

[0303] “N / A” indicates that no test was performed.

[0304] The following specific examples will be used to explain the contents of this disclosure, but the scope of this disclosure is not limited thereto.

[0305] Unless otherwise specified, the mice, proteins, and cells used in the following examples were provided by Shanghai Ruizhi Chemical Research Co., Ltd.

[0306] Example 1: Humanization and expression of D10 antibody

[0307] Humanization of mouse anti-ROR1 monoclonal antibody D10:

[0308] The mouse antibody D10 was humanized using the CDR transplantation method. The CDR region was transplanted onto the matching heavy chain variable region and light chain variable region framework sequences to obtain the heavy chain variable region sequence (as shown in SEQ ID NO:1) and light chain variable region sequence (as shown in SEQ ID NO:2) of the humanized antibody BRHu-3.

[0309] BRHu-3-VH (SEQ ID NO:1):

[0310] BRHu-3-VL (SEQ ID NO:2):

[0311] D10-VH (SEQ ID NO:3):

[0312] D10-VL (SEQ ID NO:4):

[0313] Table 1. CDR region of antibody D10 / BRHu-3

[0314] The VH and VL of the above-mentioned antibody BRHu-3, and the VH and VL of the D10 monoclonal antibody, were combined with the constant regions of the human heavy chain and κ light chain of IgG1, respectively, to obtain the humanized antibody BRHu-3 and the chimeric antibody ch-D10.

[0315] IgG1 heavy chain constant region (SEQ ID NO:11):

[0316] κ light chain constant region (SEQ ID NO:12):

[0317]

[0318] The chimeric antibody ch-D10 (heavy chain amino acid sequence as shown in SEQ ID NO:15, light chain amino acid sequence as shown in SEQ ID NO:16) and the humanized antibody BRHu-3 (heavy chain amino acid sequence as shown in SEQ ID NO:13, light chain amino acid sequence as shown in SEQ ID NO:14) were expressed and purified in CHO-K1 cells. First, the vector containing the antibody heavy and light chain coding sequences was transferred into CHO-K1 cells by electroporation. After culturing at 37°C and 5% CO2 for 4 days, the cell culture supernatant was centrifuged at 3000 rpm for 10 min. The supernatant was collected and purified with protein A to obtain the antibody (purity >95%).

[0319] BRHu-3 heavy chain sequence (SEQ ID NO:13):

[0320] BRHu-3 light chain sequence (SEQ ID NO:14):

[0321] Heavy chain sequence of chimeric antibody ch-D10 (SEQ ID NO:15):

[0322] The light chain sequence of the chimeric antibody ch-D10 (SEQ ID NO:16):

[0323] The humanized antibody BRHu-3 was subjected to affinity maturation to obtain antibodies YR-4, YR-8, YR-10, YR-11 and YR-21.

[0324] The variable region and CDR region of the antibody are shown below:

[0325] Table 2. CDR region of antibody YR-8

[0326] The variable region sequence of the antibody obtained through affinity maturation:

[0327] YR-8-VH (SEQ ID NO:22):

[0328] YR-8-VL (SEQ ID NO:23):

[0329] YR-4-VH (SEQ ID NO:24):

[0330] YR-4-VL (SEQ ID NO:25):

[0331] YR-10-VH (SEQ ID NO:26):

[0332] YR-10-VL (SEQ ID NO:27):

[0333] YR-11-VH (SEQ ID NO:28):

[0334] YR-11-VL (SEQ ID NO:23):

[0335] YR-21-VH (SEQ ID NO:29):

[0336] YR-21-VL(SEQ ID NO:23):

[0337] The VH and VL sequences described above were combined with the constant regions of the human heavy chain and κ light chain of IgG1 (as shown in Example 1) to obtain the full-length sequence of the antibody. The antibody was expressed and purified according to the method described in Example 1. The heavy and light chains of the exemplary antibody YR-8 are shown in SEQ ID NO:30 and SEQ ID NO:31, respectively.

[0338] Heavy chain sequence of YR-8 (SEQ ID NO:30):

[0339] The light chain sequence of YR-8 (SEQ ID NO:31):

[0340] Amino acid mutations were performed at positions 60 (N) and 61 (S) of the heavy chain of the YR-8 molecule (amino acid positions were determined based on Kabat numbering). Mutation schemes included: mutating N at position 60 to E, or mutating S at position 61 to A, V, T, L, and I, respectively. The resulting molecules were named YR-8-ES, YR-8-NA, YR-8-NV, YR-8-NT, YR-8-NL, and YR-8-NI. The HCDR2 and VH sequences of the antibodies obtained after mutation are as follows:

[0341] Table 3. HCDR2 sequence of YR-8 modified antibody

[0342] The VH sequence of the mutated antibody is as follows:

[0343] YR-8-ES-VH (SEQ ID NO:38):

[0344] YR-8-NA-VH (SEQ ID NO:39):

[0345] YR-8-NV-VH (SEQ ID NO:40):

[0346] YR-8-NT-VH (SEQ ID NO:41):

[0347] YR-8-NL-VH (SEQ ID NO:42):

[0348] YR-8-NI-VH (SEQ ID NO:43):

[0349] The mutated VH was combined with YR-8-VL (SEQ ID NO:23) and the constant regions of the human IgG1 heavy chain and κ light chain (as shown in Example 1) to obtain a complete antibody. The above antibody mutant was expressed and purified according to the method described in Example 1. The heavy chain and light chain of the exemplary antibody YR-8-ES obtained in this disclosure are shown in SEQ ID NO:44 and SEQ ID NO:31, respectively.

[0350] Heavy chain sequence of YR-8-ES (SEQ ID NO:44):

[0351] The light chain sequence of YR-8-ES (SEQ ID NO:31):

[0352] Example 2: Humanization of H10 antibody

[0353] The CDR region was transplanted into the mouse monoclonal antibody H10 against ROR1 using a CDR transplantation method. The CDR region was transplanted into the matching heavy chain variable region and light chain variable region framework sequences to obtain different humanized antibodies. The variable region and CDR region sequences of the humanized antibodies are shown below:

[0354] Table 4. CDR regions of H10 / H10 humanized antibodies

[0355] H10 VH (SEQ ID NO:51):

[0356] H10 VL (SEQ ID NO:52):

[0357] H10-hVH1 (SEQ ID NO:53):

[0358] H10-hVH2 (SEQ ID NO:54):

[0359] H10-hVH3 (SEQ ID NO:55):

[0360] H10-hVH4 (SEQ ID NO:56):

[0361] H10-hVL1 (SEQ ID NO:57):

[0362] H10-hVL2 (SEQ ID NO:58):

[0363] H10-hVL4 (SEQ ID NO:59):

[0364] H10-hVL5 (SEQ ID NO:60):

[0365] The variable region of the humanized H10 antibody was combined with the constant regions of the human IgG1 heavy chain and κ light chain (as shown in Example 1) to obtain a complete antibody. The specific antibody is as follows:

[0366] Table 5. H10 Humanized Antibodies

[0367] Six CDR regions of H10-hVH4-hVL4 were subjected to saturation mutagenesis. The mutated vector containing the antibody heavy and light chain coding sequences was then transferred into CHO-K1 cells via electroporation. Cells were cultured at 37°C and 5% CO2 for 4 days. The supernatant was added to microplates coated with ROR1 (manufacturer: Kaika Biotechnology, catalog number: ROR-HM401) and incubated at 37°C for 1 hour. After washing, HRP-labeled goat anti-human IgG (manufacturer: Jackson, 109-005-008) was added, and the reaction was incubated at 37°C for 1 hour. After washing, TMB solution was added, and the reaction was incubated at room temperature in the dark for 15 minutes. The reaction was then terminated by adding ELISA Stopping Solution. The absorbance was measured at 450 nm using a microplate reader. Six clones with high affinity were selected for sequencing, and the resulting six clones were named AM3-ZH1, AM3-ZH3, AM3-ZH4, AM3-ZH5, AM3-ZH6, and AM3-ZH7, respectively. The relevant antibody sequences are as follows:

[0368] Table 6. CDR sequences of H10 affinity maturation antibodies

[0369] AM3-ZH1-VH (SEQ ID NO:70):

[0370] AM3-ZH1-VL (SEQ ID NO:71):

[0371] AM3-ZH3-VH (SEQ ID NO:72):

[0372] AM3-ZH3-VL (SEQ ID NO:71):

[0373] AM3-ZH4-VH (SEQ ID NO:73):

[0374] AM3-ZH4-VL (SEQ ID NO:71):

[0375] AM3-ZH5-VH (SEQ ID NO:74):

[0376] AM3-ZH5-VL (SEQ ID NO:75):

[0377] AM3-ZH6-VH (SEQ ID NO:74):

[0378] AM3-ZH6-VL (SEQ ID NO:76):

[0379] AM3-ZH7-VH (SEQ ID NO:74):

[0380] AM3-ZH7-VL (SEQ ID NO:71):

[0381] The variable region described above is combined with the constant regions of the human IgG1 heavy chain and κ light chain (as shown in Example 1) to obtain a complete antibody. The heavy chain and light chain sequences of the exemplary antibody AM3-ZH3 obtained in this disclosure are shown in SEQ ID NO:77 and SEQ ID NO:78, respectively.

[0382] Heavy chain sequence of AM3-ZH3 (SEQ ID NO:77):

[0383] The light chain sequence of AM3-ZH3 (SEQ ID NO:78):

[0384] Example 3: Construction of a dual epitope antibody targeting ROR1

[0385] In this embodiment, a bispecific antibody targeting ROR1 was constructed using ROR1 antibodies derived from D10 and ROR1 antibodies derived from H10, wherein the ROR1 antibodies derived from D10 and ROR1 antibodies derived from H10 bind to different epitopes of ROR1, respectively.

[0386] This embodiment constructs a dual epitope antibody with a KIH structure, and an exemplary structure is shown below:

[0387] Structure 1 contains the following four chains:

[0388] Chain 1: YR8VH-CH1-Fcknob;

[0389] Chain 2: YR8VL-CL

[0390] Chain 3: AM3-ZH3VH-CH1-FcHole

[0391] Chain 4: AM3-ZH3VL-CL

[0392] Structure 2 contains the following four chains:

[0393] Chain 1: YR8VH-CH1-Fchole;

[0394] Chain 2: YR8VL-CL

[0395] Chain 3: AM3-ZH3VH-CH1-Fcknob

[0396] Chain 4: AM3-ZH3VL-CL

[0397] CH1 (SEQ ID NO:88):

[0398] The sequence of Fcknob (T366W) (SEQ ID NO:79):

[0399] The sequence of FcHole (T366S, L368A, Y407V) (SEQ ID NO:80):

[0400] To further prepare antibodies binding to different epitopes of ROR1, this disclosure also discloses the preparation of YR-8-ES variant 1, with the Fc region containing only the Knob (T366W) mutation, based on the YR-8-ES antibody, and the preparation of AM3-ZH3 variant 1, with the Fc region containing only the Hole (T366S, L368A, Y407V) mutation, based on the AM3-ZH3 antibody. The sequences are as follows:

[0401] YR-8-ES variant 1 heavy chain (SEQ ID NO:81):

[0402] YR-8-ES variant 1 light chain (SEQ ID NO:31):

[0403] AM3-ZH3 variant 1 heavy chain (SEQ ID NO:83):

[0404] AM3-ZH3 variant 1 light chain (SEQ ID NO:78):

[0405] For example, YR-8-ES antibody variant 1 and AM3-ZH3 antibody variant 1 were dissolved separately in PBS at pH 7.2, mixed in a 1:1 molar ratio, 2 mM EDTA was added, and the antibody was reduced with tris(2-carboxyethyl)phosphine hydrochloride (the molar ratio of tris(2-carboxyethyl)phosphine hydrochloride to antibody was 10:1). After incubation at 25°C for approximately 16 hours, purification and desalting were performed by elution with G25 resin, and the mixture was filtered through a 0.2 μm filter under aseptic conditions to obtain the ROR1-targeting biepisode antibody KIH YR8 / ZH3, the sequence of which is as follows:

[0406] Chain 1 of KIH YR8 / ZH3 (SEQ ID NO:81):

[0407] Chain 2 of KIH YR8 / ZH3 (SEQ ID NO:82):

[0408] Chain 3 of KIH YR8 / ZH3 (SEQ ID NO:83):

[0409] Chain 4 of KIHYR8 / ZH3 (SEQ ID NO:84):

[0410] In addition, this disclosure uses UC961 as a positive control, the sequence of which is shown below:

[0411] UC961 heavy chain (SEQ ID NO:85):

[0412] UC961 light chain (SEQ ID NO:86):

[0413] The amino acid sequence of human ROR1 (SEQ ID NO:87):

[0414] Example 4: Preparation of drug linkers

[0415] Drug linkers (LDs) are prepared by pre-coupling toxins (drugs) used to prepare ADCs with linkers; exemplary toxins include MMAE, Eribulin, and Exatecan.

[0416] In this embodiment, experimental methods without specific conditions are generally performed under standard conditions or as recommended by the raw material or product manufacturer. Reagents without a specific source are commercially available, standard reagents.

[0417] The structure of the compound was determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shifts (δ) were expressed in 10⁻¹⁰ ohms. -6 The unit (ppm) is given. 1 The H NMR measurements were performed using a Bruker AVANCE-400 NMR spectrometer, with deuterated dimethyl sulfoxide as the solvent and tetramethylsilane (TMS) as the internal standard.

[0418] MS measurements were performed using a Shimadzu LCMS-2020 Single Quadrupole LC-MS / MS system (manufacturer: Shimadzu, MS model: 2020 Single Quadrupole MS); column: Phenomenex Gemini NX 5μm, C18. 50×4.6mm, mobile phase: 0.1% aqueous solution of formic acid / 0.1% acetonitrile (ACN) solution of formic acid, flow rate: 1mL / min.

[0419] The RP-HPLC used a Shimadzu Nexera preparative liquid chromatography system, with a Phenomenex Gemini NX 5μm, C18 column. 150×50mm, mobile phase: 0.1% aqueous solution of trifluoroacetic acid / 0.1% acetonitrile (ACN) solution of trifluoroacetic acid, flow rate: 50mL / min.

[0420] 4.1 Preparation of LD-1

[0421] To 3 mL of anhydrous tetrahydrofuran containing a solution of 2-tert-butylhydrazine-1,2-dicarboxylic acid ester (76.8 mg, 0.33 mmol) and tert-butyl 3-bromo-2-(bromomethyl)propionate (200 mg, 0.66 mmol), NaH (60%, 80 mg, 2.0 mmol) was added. The mixture was stirred at room temperature for 15 minutes, and then the reaction was quenched with 1 mL of water containing 60 μL of AcOH. The mixture was then purified by RP-HPLC. The purified fraction was lyophilized to give 204 mg of a white solid, Al.

[0422] MS m / z: 373.6 [M+H] + .

[0423] To 8.0 mL of acetic acid (AcOH) solution containing tert-butyl 2-[[tert-butyloxycarbonyl-(tert-butyloxycarbonylamino)amino]methyl]prop-2-enoate (A1) (204 mg, 0.55 mmol), 3,4-dibromofuran-2,5-dione (140 mg, 0.55 mmol) was added. The mixture was refluxed and stirred under argon atmosphere for 11 days, then concentrated to 3 mL and purified by RP-HPLC to give 43 mg of white solid A2 (yield: 22%).

[0424] MS m / z: 354.8 [M+H] + .

[0425] To a stirred solution of 6,7-dibromo-5,8-dioxo-2,3,5,8-tetrahydro-1H-pyrazolo[1,2-a]pyridazine-2-carboxylic acid (A2, 350 mg) in anhydrous dichloromethane (10 mL), N-hydroxysuccinimide (230 mg) was added, followed by N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (400 mg). The mixture was stirred at room temperature for 30 minutes, and the reaction was concentrated to dryness under reduced pressure. The residue was purified directly by RP-HPLC and lyophilized to give compound 4 as a white solid (337 mg).

[0426] MS m / z: 452.0 [M+H] + .

[0427] Compound 4 (12 mg) was added to a 1 mL DMF solution containing compound 6 (31 mg, purchased from MedChemExpress, catalog number: HY-100374), followed by 0.01 mL of DIEA. The reaction mixture was stirred at room temperature (22 °C). After 3 hours, the crude reaction mixture was purified directly by RP-HPLC and lyophilized to give compound 8 (17 mg), i.e., LD-1, as a white solid.

[0428] MS m / z: 1243.6 [M+H] + .

[0429] 4.2 Preparation of LD-4

[0430] Maleimide hexanoic acid (compound 18, 12 mg) was added to a solution of anhydrous DMF (2 mL) containing compound 6 (62 mg, TFA salt), followed by DIEA (0.02 mL) and HATU (20 mg). The reaction mixture was stirred at room temperature (22 °C). After 15 minutes, the crude reaction mixture was purified directly by RP-HPLC and lyophilized to give compound 19 (62 mg, TFA salt), i.e., LD-4, as a white solid.

[0431] MS m / z: 1316.6 [M+H] + .

[0432] 4.3 Preparation of LD-14

[0433] To an anhydrous N,N-dimethylformamide (2 mL) solution containing compound 1 (77 mg, MedChemExpress, HY-41189), iribulin (compound 2, methanesulfonate, 82 mg, MedChemExpress, HY-13442) was added, followed by N-ethyldiisopropylamine (0.035 mL). The reaction mixture was stirred at room temperature (22 °C). After 6 hours, piperidine (0.1 mL) was added, and the reaction was stirred at room temperature for 15 minutes. The reaction mixture was purified directly by RP-HPLC, and after lyophilization, gave compound 3 (110 mg, trifluoroacetate) as a white solid.

[0434] Compound 4 (20 mg, synthesized according to the method reported in PCT / US2022 / 078563) was added to a solution of N,N-dimethylformamide (2 mL) containing compound 3 (50 mg), followed by N-ethyldiisopropylamine (0.01 mL). The reaction mixture was stirred at room temperature. After 3 hours, the crude mixture was purified by RP-HPLC and lyophilized to give compound 5 (43 mg), i.e., LD-14, as a white solid.

[0435] MS m / z: 1471.6 [M+H] + .

[0436] 1HNMR (400MHz, DMSO) δ: 10.00 (s, 1H), 8.36 (d, J=8.8Hz, 1H), 8.25 (d, J=7.6Hz, 1H), 7.57 (d, J=8.4H z, 2H), 7.27 (d, J=8.4Hz, 2H), 7.08 (t, J=6Hz, 1H), 5.97 (t, J=5.6Hz, 1H), 5.41 (s, 2H), 5.05 (s, 1H), 5.00 (s, 1H), 4.97-4.89 (m, 2H), 4.83 (s, 1H), 4.75 (s, 1H), 4.63 (s, 1H), 4.55 (dd, J=7.2, 4.4Hz, 2H ), 4.38 (dd, J=13.2Hz, J=7.6Hz, 1H), 4.32–4.21 (m, 4H), 4.21–4.12 (m, 3H), 4.22-4.06 (m, 3H), 4.02 (t, J=7.6Hz, 1H), 3.86-3.73 (m, 2H), 3.73-3.62 (m, 2H), 3.58–3.45 (m, 3H), 3.30 (s, 1H), 3.28-3.1 8 (m, 4H), 3.06–2.90 (m, 4H), 2.84 (d, J=9.6Hz, 1H), 2.80-2.63 (m, 2H), 2.61-2.52 (m, 1H), 2.37–2.1 7(m, 5H), 2.17–2.08(m, 1H), 2.06–1.85(m, 7H), 1.78-1.54(m, 7H), 1.54-1.39(m, 4H), 1.39–1.26(m , 4H), 1.24-1.11 (m, 1H), 1.03 (d, J=6.4Hz, 3H), 1.01-0.92 (m, 1H), 0.86 (dd, J=12.4, J=6.8Hz, 6H).

[0437] Example 5: Preparation of ADC

[0438] The antibody disclosed herein was reacted with a drug linker (LD) to prepare an ADC drug, and the DAR value of the ADC was determined.

[0439] In simple terms, antibodies are treated with reducing agents (such as tris(2-carboxyethyl)phosphine hydrochloride (TCEP) or dithiothreitol (DTT)) to reduce some or all of the cysteine ​​disulfide residues, forming highly nucleophilic cysteine ​​thiol groups (-CH2SH). This allows the partially or completely reduced antibody to react with a drug linker, ultimately producing an ADC.

[0440] Methods for determining the DAR value of ADC:

[0441] The DAR value of the ADC disclosed herein was analyzed using hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC). The ADC was separated in the column using a MabPac HIC-Butyl analytical column (4.6 × 100 mm, 5 μm, catalog number 088558, Thermo Fisher, USA). Buffer solution A was prepared with 25 mM sodium phosphate buffer (pH 6.8) containing 1.5 M ammonium sulfate, and buffer B was prepared with 25 mM sodium phosphate buffer (pH 6.8) containing 25% acetonitrile. 85% A and 15% B were stabilized as initial conditions. Elution was performed using a linear gradient of 85% A and 15% B versus 5% A and 95% B for 30 min, followed by an additional 5 min elution using 5% A and 95% B. Flow rate and temperature were set to 0.5 mL / min and 25 °C. ADC drug distribution was detected at 214 and 280 nm for DAR value calculation.

[0442] 5.1 Preparation of ADC-1

[0443] Antibody UC961 was dissolved in PBS at pH 7.2, 2 mM EDTA was added, and the antibody was reduced with tris(2-carboxyethyl)phosphine hydrochloride (molar ratio of tris(2-carboxyethyl)phosphine hydrochloride to antibody was 2.8:1). After incubation at 37°C for approximately 120 minutes, drug linker LD-4 was added to the reduced antibody (molar ratio of drug linker to antibody was 5:1). The mixture was purified and desalted by elution with G25 resin at room temperature for 1 hour, and filtered aseptically through a 0.2 μm filter to obtain ADC-1, which was then frozen and stored. Analysis using the above hydrophobic interaction chromatography-high performance liquid chromatography (HPLC) determined that the average DAR value of ADC-1 was between 3.7 and 4.3.

[0444] 5.2 Preparation of ADC-2

[0445] 2 mM EDTA was added to antibody AM3-ZH3 dissolved in PBS at pH 7.2, and the antibody was reduced with tris(2-carboxyethyl)phosphine hydrochloride (molar ratio of tris(2-carboxyethyl)phosphine hydrochloride to antibody was 10:1). After incubation at 37°C for approximately 120 minutes, drug linker LD-14 was added to the reduced antibody (molar ratio of drug linker to antibody was 5:1). The mixture was purified and desalted by elution with G25 resin at room temperature for 1 hour, and filtered aseptically through a 0.2 μm filter to obtain ADC-2, which was then frozen and stored. Analysis using the above hydrophobic interaction chromatography-high performance liquid chromatography (HPLC) determined that the average DAR value of ADC-2 was between 3.7 and 4.3.

[0446] 5.3 Preparation of ADC-3 (Preparation of Bispecific Antibody-Drug Conjugate)

[0447] YR-8-ES antibody variant 1 and AM3-ZH3 antibody variant 1 were dissolved separately in PBS at pH 7.2 and mixed at a 1:1 molar ratio. 2 mM EDTA was added, and the antibody was reduced with tris(2-carboxyethyl)phosphine hydrochloride (tris(2-carboxyethyl)phosphine hydrochloride:antibody molar ratio was 10:1). After incubation at 25°C for approximately 16 hours, the drug linker LD-14 was added to the reduced KIHYR8 / ZH3 antibody (drug linker:antibody molar ratio was 5:1). The mixture was purified and desalted by elution with G25 resin at room temperature for 1 hour, and then filtered aseptically through a 0.2 μm filter to obtain ADC-3, which was then frozen and stored. Analysis using the above hydrophobic interaction chromatography-high performance liquid chromatography (HPLC) determined that the average DAR value of ADC-3 was between 3.7 and 4.3.

[0448] 5.4 Preparation of ADC-4

[0449] YR-8-ES antibody variant 1 and AM3-ZH3 antibody variant 1 were dissolved separately in PBS at pH 7.2 and mixed at a 1:1 molar ratio. 2 mM EDTA was added, and the antibody was reduced with tris(2-carboxyethyl)phosphine hydrochloride (tris(2-carboxyethyl)phosphine hydrochloride:antibody molar ratio 10:1). After incubation at 25°C for approximately 16 hours, drug linker LD-1 was added to the reduced KIHYR8 / ZH3 antibody (drug linker:antibody molar ratio 5:1). The mixture was purified and desalted by elution with G25 resin at room temperature for 1 hour, and filtered aseptically through a 0.2 μm filter to obtain ADC-4, which was then frozen and stored. Analysis using the above hydrophobic interaction chromatography-high performance liquid chromatography (HPLC) determined that the average DAR value of ADC-4 was between 3.7 and 4.3.

[0450] 5.5 Preparation of ADC-5

[0451] Antibody AM3-ZH3 was dissolved in PBS at pH 7.2, 2 mM EDTA was added, and the antibody was reduced with tris(2-carboxyethyl)phosphine hydrochloride (tris(2-carboxyethyl)phosphine hydrochloride:antibody molar ratio of 10:1). After incubation at 37°C for approximately 120 minutes, 5 molar ratio of drug linker LD-1 was added to the reduced antibody (LD-1:antibody molar ratio of 5:1). The mixture was purified and desalted by elution with G25 resin at room temperature for 1 hour, and filtered aseptically through a 0.2 μm filter to obtain ADC-5, which was then frozen and stored. Analysis using the above hydrophobic interaction chromatography-high performance liquid chromatography determined that the average DAR value of ADC-5 was between 3.7 and 4.3.

[0452] In addition, using the same method as that used to prepare ADC-2 and ADC-5, negative control IgG and positive control UC961 antibodies were prepared and conjugated with LD-14 and LD-1, respectively, and named ADC-6 (UC961 conjugated with LD-14) and ADC-7 (UC961 conjugated with LD-1), ADC-8 (IgG conjugated with LD-14) and ADC-9 (IgG conjugated with LD-1), respectively, with drug loadings of 3.7–4.3.

[0453] The exemplary ADCs prepared are summarized in Table 7 below:

[0454] Table 7

[0455] Example 6: Detection of biological activity of monoclonal antibodies

[0456] 6.1 Binding activity of monoclonal antibodies to human ROR1 protein

[0457] Affinity assays for ROR1 protein were performed using the OctetR8 (Sartorius) instrument. An AHC (Anti-hIgG Fc Capture) biosensor (Sartorius) was used to capture the antibody, which was then immersed in the ROR1 antigen analyte. The experiment consisted of five steps:

[0458] 1. Baseline (60 seconds), 2. Loading (antibody capture) (150 seconds, 1.5 nm), 3. Baseline (100 seconds), 4. Association (binding antigen ROR1, 60 seconds), 5. Dissociation (dissociating antigen ROR1, 60 seconds). After the test, the sensor was regenerated by alternating immersion in regeneration buffer (glycine, pH 1.5) and neutralization buffer (PBS) for 5 seconds each, for a total of 3 cycles. PBS was used as the running buffer in this experiment.

[0459] 6.2 Binding activity of monoclonal antibodies to human ROR1-expressing cells

[0460] The binding of the disclosed anti-ROR1 antibody to human ROR1-expressing cells MDA-MB-231 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) and Jeko-1 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) was detected by flow cytometry. MDA-MB-231 / Jeko-1 cells were incubated with different concentrations (starting at 66.67 nM, 6-fold dilution, for a total of 8 concentrations) of antibody at 4°C for 30 minutes. After washing the cells twice with 2% BSA-PBS, PE-labeled goat anti-human IgG Fc (Invitrogen, catalog number: 12-4998-82, 1:100 dilution) was added, and the reaction was carried out at 4°C in the dark for 30 minutes. After washing the cells twice with 2% BSA-PBS, the median fluorescence intensity was detected by flow cytometry using a BD C6 plus instrument.

[0461] 6.3 Binding activity of monoclonal antibodies to human ROR2 protein

[0462] The binding of humanized antibody to ROR1 homolog ROR2 was detected using an ELISA method. hROR2 (manufacturer: Kaika Biotechnology, catalog number: ROR-HM402) was diluted to 1 μg / mL in PBS and coated onto microplates, then incubated at 37°C for 1 hour. The plates were then blocked with 5% BSA-PBS blocking buffer at 37°C for 1 hour. After washing with PBST, the antibody was diluted to different concentrations (starting from 333.33 nM, 3-fold dilutions, for a total of 8 concentrations) and added to the plates, then incubated at 37°C for 1 hour. After washing, HRP-labeled goat anti-human IgG (manufacturer: Sigma-Aldrich, catalog number A0170, 1:10000 dilution) was added, and the reaction was carried out at 37°C for 1 hour. After washing, TMB solution was added, and the reaction was incubated at room temperature in the dark for 15 minutes, then the reaction was terminated by adding ELISA Stopping Solution. The absorbance was measured at 450 nm using a microplate reader.

[0463] The results are shown in Table 8-11 below.

[0464] Table 8. Results of activity assay for humanized antibody BRHu-3 at D10

[0465] Table 8 shows that the humanized antibody BRHu-3 and the ch-D10 antibody have similar affinity for ROR1 protein, and similar binding ability to MDA-MB-231 cells, and neither of them binds to ROR2.

[0466] Table 9. Activity assay results of affinity maturation antibodies

[0467] The results in Table 9 show that, compared with BRHu-3, the affinity of the maturation antibody YR-8 to ROR1 is significantly improved, and YR-8 does not bind to ROR2.

[0468] Table 10. Results of YR-8 mutant molecule activity assay

[0469] Table 10 shows that neither YR-8 nor YR-8-ES binds to ROR2. Compared with YR-8, YR-8-ES has comparable affinity for ROR1 and binding activity with MDA-MB-231 cells.

[0470] Table 11. Results of Affinity Maturation Molecular Activity Detection

[0471] Table 11 shows that, compared to H10-hVH4-hVL4, the affinity maturation antibody AM3-ZH3 showed varying degrees of increased affinity for ROR1 and binding to MDA-MB-231 cells, with AM3-ZH3 exhibiting an affinity increase of more than 9-fold. Furthermore, AM3-ZH3 did not bind to ROR2.

[0472] Example 7: Antibody binding experiment with ROR1-expressing cells

[0473] Measuring the specific binding ability of anti-hROR1 antibodies to the hROR1 antigen expressed on the cell surface is crucial for antibody efficacy. The cell lines used in this experiment were PA-1 (ATCC CRL-1572 human ovarian teratoma cells) and Jeko-1 (ATCC CRL-3006, human mantle cell lymphoma cells). PA-1 cells were cultured in ATCC EMEM medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. Jeko-1 cells were cultured in RPMI-1640 medium containing 20% ​​fetal bovine serum at 37°C in a 5% CO2 incubator. The experimental procedure is as follows:

[0474] Cells in logarithmic growth phase were lysed with 0.25% trypsin, resuspended in staining buffer (Biolengend), washed, counted, and adjusted to 4.5 × 10⁻⁶ cells / mL. 5Add 45 μL of staining buffer per 100 μL of cells to each well of a 96-well plate. Add 5 μL of human TruStain (BioLegend) receptor blocking agent to each well to prevent non-specific binding of the antibody to human tumor cells. Dilute the test antibody to an initial concentration of 200 nM in staining buffer, then perform 3-fold dilutions to obtain 11 concentration points, including 200 nM and zero. Add 50 μL of the diluted test antibody to each well of the 96-well plate, resulting in a final maximum concentration of 100 nM. Mix well and incubate at 4°C for 15 minutes. After the reaction, wash the cells in staining buffer, then resuspend the PE-labeled constant region (Fc) specific antibody (rabbit anti-human IgG PE conjugate, BioLegend, 410707) in 5 μL / 2×10⁻⁶ cells / well. 5 Cells were incubated at 100 μL staining buffer per cell and reacted at 4 °C for 15 min. After the reaction, cells were washed in staining buffer and resuspended in 100 μL staining buffer. Single-cell readings on the PE channel were analyzed using a Novocyte 3000 (Agilent) instrument. A dose-response curve was plotted using the Sigmoidal,4PL four-parameter equation, with antibody protein concentration on the x-axis and the corresponding PE channel reading on the y-axis. EC was generated after analysis. 50 The value is: Y = Bottom + (X^Hillslope) * (Top - Bottom) / (X^HillSlope + EC) 50 ^HillSlope),

[0475] MFI fold = highest PE reading in the antibody-treated experimental group / PE reading in the untreated group;

[0476] The FACS binding activity results of the anti-hROR1 antibody assays with human ROR1 are shown in Table 12. The results show that the anti-ROR1 antibodies disclosed herein can specifically bind to PA-1 and Jeko-1 cells expressing ROR1, and both exhibit high binding capacity. Furthermore, on the same cell line, the total amount of antigen that the dual-epitope antibody can recognize and label is higher than that of the single-epitope antibody, specifically manifested as a significant increase in the MFI fold ratio.

[0477] Table 12. Binding ability of anti-hROR1 antibody to cell lines

[0478] Example 8: Cytotoxicity assay of ADC molecules

[0479] This experiment investigated the inhibitory effects of anti-ROR1 antibody-MMAE conjugate and anti-ROR1 antibody-Eribulin conjugate on the proliferation of various tumor cell lines. CellTiterGlo2 (Promega) reagent was used to evaluate the anti-proliferative effects of the drugs. The cell lines used in the experiment were: PA-1 (ATCC, CRL-1572 human ovarian teratoma cells), Jeko-1 (ATCC CRL-3006, human mantle cell lymphoma cells), and HCC-1187 (ATCC CRL-2322 human breast cancer cells). The experimental procedure is as follows:

[0480] PA-1 cells were cultured in ATCC EMEM medium containing 10% fetal bovine serum, Jeko-1 cells in RPMI-1640 medium containing 20% ​​fetal bovine serum, and HCC-1187 cells in RPMI-1640 medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. The three cell types were cultured at 2 × 10⁻⁶ cells / mL. 3 ~5×10 3 Cells were seeded at a density of 50 μL / well in 96-well plates. After 24 h of culture, 100 μL / well of anti-ROR1 antibody-MMAE conjugate, anti-ROR1 antibody-Eribulin conjugate, or control antibody-MMAE conjugate or control antibody-Eribulin conjugate diluted with different concentrations of culture medium was added. Each concentration was replicated, and corresponding concentrations of solvent control and cell-free culture medium were also included. After culturing at 37°C and 5% CO2 for 96 h (PA-1, Jeko-1) or 144 h (HCC-1187), 100 μL of CellTiterGlo2 was added to each well. The plates were mixed at room temperature on a shaker for 15 min, and the luminescence value was measured. The IC50 of the anti-ROR1 antibody-MMAE conjugate or anti-ROR1 antibody-Eribulin conjugate for various cell types was calculated. 50 Value (nM). The results are shown in Table 13.

[0481] Table 13. IC50 of ADCs for inhibiting different cell proliferation 50 Value (nM)

[0482] As shown in Table 13, the anti-ROR1 antibody-MMAE conjugate or the -Eribulin conjugate exhibited significant killing effects on tumor cells with different ROR1 expression levels. The inhibitory effect was related to the different conjugation methods or different toxins used. This indicates that the anti-ROR1 antibody conjugate disclosed herein can specifically kill ROR1-positive cells through ROR1-mediated endocytosis. Furthermore, it showed significant inhibitory effects on cancer cell lines from breast cancer, ovarian cancer, and lymphoma, respectively, demonstrating that the ADC obtained in this disclosure has good inhibitory effects on various solid tumors and hematological malignancies.

[0483] Example 9: In vivo efficacy testing of ADC molecules

[0484] 9.1 ADC Efficacy Testing in a Mouse Model Transplanted with HCC1187 Breast Cancer Cell Line

[0485] 1×10 7 One cell / human ROR1-expressing breast cancer cell line HCC1187 (purchased from ATCC) was transplanted subcutaneously into female severely combined immunodeficient CB17 SCID mice (purchased from Charles River Laboratories). Following transplantation, when the tumor size reached an average of 162 mm... 3 On day 0, mice were divided into groups and administered either a single intravenous injection of 3.0 mg / kg ADC1, ADC2, ADC3, ADC4, or ADC5 prepared in Example 8, or a total of two intravenous injections of 1.0 mg / kg ADC2, ADC3, ADC4, or ADC5 every four days (day 0 and day 4, Q4D×2). In the control group, mice were administered 4 mL / kg PBS intravenously. For the next 48 days, tumor size and body weight were measured, and tumor inhibition rate (TGI) was calculated. The TGI was calculated as follows: TGI(%) = 1 - [(Td-T0) / (Cd-C0)] × 100%

[0486] Where Td and Cd are the average tumor volumes of the treatment group and the control group on the day of tumor volume measurement, and T0 and C0 are the average tumor volumes of the treatment group and the control group on day 0.

[0487] The results are shown in Figure 1 and Table 14.

[0488] Table 14. Tumor inhibition rate of different ADCs on subcutaneous tumors in HCC1187 mice

[0489] The results showed that the ADC disclosed in this study could significantly inhibit the growth of HCC1187 xenografts in mice.

[0490] 9.2 ADC Efficacy Testing in a Jeko-1 Cellular Lymphoma Cell Line Transplanted Mouse Model

[0491] 1×10 7 Jeko-1 mantle cell lymphoma cells (purchased from ATCC) expressing only human ROR1 were transplanted subcutaneously into female severely combined immunodeficient CB17 SCID mice (purchased from Charles River Laboratories). Following transplantation, tumors reached an average size of 156 mm. 3On day 0, mice were divided into groups and administered either a single intravenous injection of 3.0 mg / kg ADC1, ADC2, ADC3, ADC4, or ADC5 prepared in Example 8, or a total of three intravenous injections of 1.0 mg / kg ADC2, ADC3, ADC4, or ADC5 every four days (day 0, day 4, and day 8, Q4D×3). In the control group, mice were administered 4 mL / kg PBS intravenously. After 51 days, tumor size and body weight were measured, and tumor inhibition rate (TGI) was calculated. Results are shown in Figure 2 and Table 15.

[0492] Table 15. Tumor inhibition rate of different ADCs on subcutaneous tumors in Jeko-1 mice

[0493] The results showed that the ADC disclosed in this study could significantly inhibit the growth of Jeko-1 xenografts in mice.

[0494] 9.3 ADC Efficacy Testing in a Mouse Model with PA-1 Ovarian Cancer Cell Line Transplantation

[0495] 5×10 6 One cell / one human ROR1-expressing ovarian cancer cell line PA-1 (purchased from ATCC) was transplanted subcutaneously into female nude mice (purchased from Charles River Laboratories). Following transplantation, when the tumor size reached an average of approximately 150 mm... 3 On day 0, mice were divided into groups and administered a single intravenous injection of 1.0 mg / kg or 3.0 mg / kg ADC1 or ADC3 prepared in Example 8. In the control group, mice were administered 4 mL / kg PBS intravenously. Tumor size and body weight were measured and TGI were calculated over 13 days. Results are shown in Figure 3 and Table 16.

[0496] Table 16. Tumor inhibition rate of different ADCs on subcutaneous tumors in PA-1 mice

[0497] The results showed that the ADC disclosed in this study could significantly inhibit the growth of PA-1 xenografts in mice.

[0498] Example 10. Screening of stable formulations of anti-ROR1 antibody-drug conjugates

[0499] Different component solutions of the anti-ROR1 antibody-drug conjugate were prepared using water for injection. After lyophilization using process 1 (see Example 11), each formulation was subjected to stability testing. The experimental conditions for the stability testing are shown in Table 17 below. The anti-ROR1 antibody-drug conjugate mentioned above is ADC-3.

[0500] Table 17. Experimental conditions for stability testing

[0501] Reagents and testing instruments

[0502] Reagents:

[0503] Sucrose (purchased from Merck KGaA), polysorbate 20 (purchased from Croda), citric acid (purchased from Merck KGaA), sodium citrate (purchased from Merck KGaA), sodium hydroxide (purchased from Hunan Ercon Pharmaceutical Co., Ltd.), and mannitol (purchased from Merck KGaA).

[0504] instrument:

[0505] Agilent 1200 liquid chromatograph (purchased from Agilent Technologies, USA) and Agilent 7100 capillary electrophoresis system (purchased from Agilent Technologies, USA).

[0506] Experimental methods

[0507] SEC-HPLC method: The chromatographic column was an Ankylo SEC-300 from SHIMSEN, 7.8 × 300 mm; the mobile phase contained 0.0043 mol / L sodium dihydrogen phosphate dihydrate, 0.0117 mol / L anhydrous disodium hydrogen phosphate, and 0.425 mol / L sodium chloride buffer, pH 7.4 ± 0.02; the flow rate was 0.5 mL / min; the column temperature was 25 ℃; the run time was 35 min; and the wavelength was 280 nm.

[0508] Non-reducing CE-SDS method: The instrument was an Agilent 7100 capillary electrophoresis system; the effective capillary length was 85 mm and the total length was 310 mm; the injection time was 5 kV for 30 s; the operating voltage was 15.0 kV; the running time was 20 min; the detection wavelength was 220 nm; and the column temperature was 25 ℃.

[0509] iCIEF method:

[0510] The catholyte was 100 mM NaOH (containing 0.1% methylcellulose solution), and the anolyte was 80 mM H3PO4 (containing 0.1% methylcellulose solution). The focusing voltage and time used were 1.5 kV for 1 min followed by 3 kV for 10 min. The preparation method of the injection solution is shown in Table 18.

[0511] Table 18

[0512] HIC-HPLC method:

[0513] The chromatographic column was TOSOH TSKgel HIC-ADC Butyl, 4.6×100mm, 5μm; the flow rate was 0.8mL / min; the column temperature was maintained at 25℃; the injection volume was 10μL; the sample concentration was 4mg / mL; the detection wavelength was 280nm; mobile phase A was 1.5mol / L ammonium sulfate, 0.026mol / L sodium dihydrogen phosphate dihydrate, pH 6.8±0.02; mobile phase B was 0.026mol / L sodium dihydrogen phosphate dihydrate, 25% isopropanol; gradient elution was run for 30min according to the gradient in Table 19 below, where A (%) and B (%) refer to the volume percentages of phase A and phase B, respectively.

[0514] Table 19

[0515] ADC content detection method:

[0516] Protein content was determined using a Shimadzu UV-2000 spectrophotometer. The concentrations of the drug and antibody were determined by measuring the absorbance of the sample at 248 nm and 280 nm, respectively, and by calculating the extinction coefficients of the antibody and drug at these wavelengths. In this disclosure, the concentration of the antibody protein is used to represent the concentration of the ADC.

[0517] 10.1. pH Screening of Anti-ROR1 Antibody-Drug Conjugate Formulations

[0518] The anti-ROR1 antibody-drug conjugate was subjected to ultrafiltration, replacing the original buffer solution in the conjugate with 20 mM sodium citrate. The ADC (anti-ROR1 antibody-drug conjugate) concentration was determined, and the following formulation was prepared:

[0519] Table 20. Formulation composition for pH screening experiments

[0520] The above-mentioned prescription preparations were prepared as solutions, and after being freeze-dried by process 1 (see Example 11), they were stored at 5℃±3℃ as samples at 0℃. High temperature test, light test and accelerated test were carried out according to Table 17. The appearance, visible foreign matter and purity (SEC-HPLC, non-reducing CE-SDS, icIEF, HIC-HPLC) of the preparation combinations F1 to F3 were detected.

[0521] The appearance and visible foreign matter of the freeze-dried product are as follows:

[0522] None of the prescriptions had obvious appearance defects; they were all white or off-white lumps.

[0523] The test results after reconstitution are as follows:

[0524] The lyophilized formulations of combinations F1 to F3 were reconstituted with sterile water for injection. The composition of the reconstituted solution is shown in Table 20.

[0525] Under conditions of 0°C, high temperature, light irradiation, and accelerated testing, no visible foreign matter was found in any of the reconstituted solution samples of formulation combinations F1 to F3.

[0526] The purity of the reconstituted solutions of formulations F1–F3 was determined by SEC-HPLC, non-reducing CE-SDS, icIEF, and HIC-HPLC. The results are as follows:

[0527] SEC-HPLC detection results:

[0528] Table 21. SEC-HPLC Detection Results

[0529] SEC-HPLC results showed that under high-temperature conditions, there were no significant changes in the composition of each formulation. Under light irradiation conditions, the main peak of each formulation decreased slightly, and the polymer content increased slightly, but the changes were minor, while the fragment peak content remained basically stable. Under accelerated irradiation conditions, the purity of the main peak, polymer content, and fragment peak content of each formulation showed no significant changes. These results fully demonstrate that the disclosed formulation combinations F1–F3 exhibit excellent stability under high-temperature, light irradiation, and accelerated irradiation conditions.

[0530] Non-reducing CE-SDS test results:

[0531] Table 22. Results of Non-Reducing CE-SDS Detection

[0532] Non-reducing CE-SDS results showed that the contents of main peak 1, main peak 2, and main peak 1 + main peak 2 of each formulation remained minimally changed during high-temperature and accelerated testing. This result demonstrates the good stability of the disclosed formulation under various environmental stress conditions.

[0533] iCIEF test results:

[0534] Table 23. iCIEF Detection Results

[0535] Note: N / A indicates not detected.

[0536] iCIEF results showed that the isoelectric point of the main peak of each formulation remained unchanged in the accelerated test, which fully demonstrates that the formulation disclosed herein has good stability under harsh conditions such as high temperature, light exposure and accelerated testing.

[0537] HIC-HPLC detection results:

[0538] Table 24. HIC-HPLC Detection Results

[0539] Note: 0.0 means that the content of naked antibody is 0, that is, no naked antibody was detected.

[0540] HICC-HPLC results showed that under high temperature, light irradiation, and accelerated testing conditions, the DAR values ​​and naked antibody ratios of each formulation did not change significantly. This indicates that the disclosed formulation has excellent stability.

[0541] In summary, the lyophilized formulations F1–F3 of the anti-ROR1 antibody-drug conjugates disclosed herein exhibit good appearance and demonstrate excellent stability under various conditions, including high temperature, light exposure, and accelerated testing. For example, aggregate content, charge distribution, and drug-antibody ratio remain stable. Therefore, citrate-sodium citrate buffer can be used as a buffer for the antibody-drug conjugates disclosed herein, with a pH range of 5.5–6.5.

[0542] 10.2 Stabilizer Screening for Anti-ROR1 Antibody-Drug Conjugate Formulations

[0543] The anti-ROR1 antibody-drug conjugate was subjected to ultrafiltration, replacing the original buffer solution in the conjugate with 20 mM sodium citrate. The ADC (anti-ROR1 antibody-drug conjugate) concentration was determined, and the following formulation was prepared:

[0544] Table 25. Formulation Composition

[0545] The above-mentioned prescription formulations were prepared as solutions, and after being freeze-dried by process 1 (see Example 11), they were stored at 5℃±3℃ as samples at 0℃. High temperature, light exposure and accelerated testing were carried out according to Table 17. The appearance, visible foreign matter and purity (SEC-HPLC, non-reducing CE-SDS, icIEF, HIC-HPLC) of formulation combinations F2 and F4 were detected.

[0546] The appearance and visible foreign matter of the freeze-dried product are as follows:

[0547] None of the prescriptions had obvious appearance defects; they were all white or off-white lumps.

[0548] The test results after reconstitution are as follows:

[0549] The lyophilized formulations of combinations F2 and F4 were reconstituted with sterile water for injection. The composition of the reconstituted solution is shown in Table 25.

[0550] The results showed that no visible foreign matter was found in any of the reconstituted solution samples of formulations F2 and F4 under conditions of 0°C, high temperature, light irradiation, and accelerated testing.

[0551] The purity of the reconstituted solutions of formulations F2 and F4 was determined by SEC-HPLC, non-reducing CE-SDS, iC IEF, and HIC-HPLC. The results are as follows:

[0552] Table 26. Stability Results

[0553] Note: 0.0 means the content of naked antibody is 0, that is, the presence of naked antibody was not detected; N / A means not detected.

[0554] The results show:

[0555] Under high temperature, light, and accelerated testing conditions, both formulations F2 and F4 showed good stability, with F2 exhibiting a higher main peak content, slightly better than F4.

[0556] Therefore, the stabilizer can be sucrose or a mixture of sucrose and mannitol.

[0557] Example 11. Optimization of the lyophilization process for anti-ROR1 antibody-drug conjugate formulations

[0558] The antibody-drug conjugate (ADC-3) was mixed with a buffer, stabilizer, and surfactant to obtain a liquid formulation; the components of the liquid formulation are shown in Table 27 below.

[0559] Table 27. Liquid Formulation Components of Anti-ROR1 Antibody-Drug Conjugates

[0560] The above-mentioned liquid formulations were freeze-dried in a lyophilizer (Martin Christ Epsilon 2-6D LSCplus) using processes 1, 2, and 3, respectively. The freeze-drying steps included pre-cooling, pre-freezing, primary drying, and secondary drying in sequence. Process 2 reduced the pre-freezing and annealing step compared to Process 1 to evaluate the impact on ice crystal formation and drying efficiency. Process 3 adjusted the primary drying stage compared to Process 2 to explore the potential impact of shortened drying time on product quality. The parameters and experimental procedures for processes 1, 2, and 3 are shown in Table 28 below.

[0561] Table 28. Freeze-drying process parameters

[0562] After obtaining the lyophilized formulation, it was stored at 5℃±3℃, and the lyophilized samples underwent visual inspection. The product was analyzed by weighing to determine the amount of water removed from each sample.

[0563] The results showed that the freeze-drying process effectively removed moisture. Products from freeze-dried processes 1 and 2 exhibited good consistency and no obvious defects, while the product from freeze-dried process 3 showed signs of spraying. Therefore, freeze-drying processes 1 and 2 are both suitable for freeze-drying anti-ROR1 antibody-drug conjugates. Freeze-drying process 2, which eliminates the annealing and warming step, has a shorter freeze-drying time and does not involve warming, making it a superior freeze-drying process.

Claims

1. A pharmaceutical composition comprising an anti-ROR1 antibody-drug conjugate, a buffer, a stabilizer, and a surfactant; wherein, The anti-ROR1 antibody comprises a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain comprises a heavy chain variable region VH1 and a light chain variable region VL1, and the second antigen-binding domain comprises a heavy chain variable region VH2 and a light chain variable region VL2, wherein: The heavy chain variable region VH1 includes HCDR1 as shown in SEQ ID NO:17, HCDR2 as shown in SEQ ID NO:32, and HCDR3 as shown in SEQ ID NO:19; and the light chain variable region VL1 includes LCDR1 as shown in SEQ ID NO:20, LCDR2 as shown in SEQ ID NO:21, and LCDR3 as shown in SEQ ID NO:10; and the heavy chain variable region VH2 includes HCDR1 as shown in SEQ ID NO:61, HCDR2 as shown in SEQ ID NO:46, and HCDR3 as shown in SEQ ID NO:65; and the light chain variable region VL2 includes LCDR1 as shown in SEQ ID NO:63, LCDR2 as shown in SEQ ID NO:64, and LCDR3 as shown in SEQ ID NO:

50. The drug is Eribulin, which is linked to an anti-ROR1 antibody via a linker.

2. The pharmaceutical composition according to claim 1, wherein the anti-ROR1 antibody comprises: The heavy chain variable region VH1 contains the amino acid sequence shown in SEQ ID NO:38, or contains a sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:38; the light chain variable region VL1 contains the amino acid sequence shown in SEQ ID NO:23, or contains a sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:23; the heavy chain variable region VH2 contains the amino acid sequence shown in SEQ ID NO:72, or contains a sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:72; and the light chain variable region VL2 contains the amino acid sequence shown in SEQ ID NO:71, or contains a sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:

71. Preferably, the anti-ROR1 antibody comprises four chains as shown in (a)-(d) below: (a) [heavy chain variable region VH1]-[CH1]-[Fc1], (b) [Light chain variable region VL1]-[CL1], (c)[heavy chain variable region VH2]-[CH1]-[Fc2], and (d)[Light chain variable region VL2]-[CL2]; Alternatively, the anti-ROR1 antibody may comprise the four chains shown in (e), (b), (f), and (d) below: (e)[heavy chain variable region VH1]-[CH1]-[Fc2], (b) [Light chain variable region VL1]-[CL1], (f)[heavy chain variable region VH2]-[CH1]-[Fc1], and (d)[Light chain variable region VL2]-[CL2]; in, The structures shown in equations (a), (b), (c), (d), (e), and (f) are arranged from the N-terminus to the C-terminus; CL1 and CL2 are each independently a light chain constant region of the antibody, CH1 is the first part of the heavy chain constant region of the antibody, and Fc1 and Fc2 are subunits of antibody Fc, wherein Fc1 has a knotted structure according to the knife-in-hole technique, and Fc2 has a hole structure according to the knife-in-hole technique; more preferably, The anti-ROR1 antibody comprises the following four chains: Chain 1, comprising an amino acid sequence as shown in SEQ ID NO:81, or comprising a sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:81; Chain 2, comprising an amino acid sequence as shown in SEQ ID NO:82, or comprising a sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:82; Chain 3, comprising an amino acid sequence as shown in SEQ ID NO:83, or comprising a sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:83; and Chain 4, comprising the amino acid sequence shown in SEQ ID NO:84, or comprising a sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:84; or The anti-ROR1 antibody comprises the following four chains: Chain 1, comprising an amino acid sequence of positions 1-446 as shown in SEQ ID NO:81, or comprising a sequence having at least 85% sequence identity with the amino acid sequence of positions 1-446 shown in SEQ ID NO:81; Chain 2, comprising an amino acid sequence as shown in SEQ ID NO:82, or comprising a sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:82; Chain 3, comprising an amino acid sequence of positions 1-447 as shown in SEQ ID NO:83, or comprising a sequence having at least 85% sequence identity with the amino acid sequence of positions 1-447 shown in SEQ ID NO:83; and Chain 4, comprising an amino acid sequence as shown in SEQ ID NO:84, or comprising a sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:

84.

3. The pharmaceutical composition according to claim 1, wherein the anti-ROR1 antibody-drug conjugate has the following structure: Where Ab is an anti-ROR1 antibody; L is an enzyme-cleavable linker, and the linker forms a bond with the sulfur atom of the anti-ROR1 antibody; D is the drug erribulin; and n is an integer or decimal from 1 to 10.

4. The pharmaceutical composition according to claim 3, wherein the L has a -L a -L b -L c -L d - structure, L a As an extension unit, L b L is a spacer unit or does not exist. c If it is an oligopeptide unit or not present, L d For spacer units or L d It does not exist, and L a Linked to antibodies, L d Connected to a drug; preferably, i)L a for L b For -C(O)- or -C(O)-NH-(CH2)2-C(O)-, L c For -glycine-glycine-phenylalanine-glycine-, -valine-citrulline-, -glycine- or bonds, and L d for -NH-CH2-O-CH2-C(O)- or bond; or ii)L a for L b The expression is -(CH2)mC(O)-, where m is 2 or 5, and L c For -valine-citrulline- or -glycine-, and L d for or iii)L a for L b For -NH-(CH2-CH2-O)4-(CH2)2-C(O)-, -NH-(CH2)2-C(O)- or bonds, L c For -valine-citrulline-, -glycine-, -glycine-glycine-phenylalanine-glycine- or bonds, and L d for Or -NH-CH2-O-CH2-C(O)-; and iiii)L a for L b For -(CH2)3-C(O)-, L c For -valine-citrulline-, and L d for in, wavy lines * indicates the connection point with Ab, and * indicates the connection point with L. b The connection point, a* represents the connection with L. c The connection point is represented by b*, where b* represents the connection point with the drug. Preferably, the L has the following structure: Where 1 represents the binding site with the anti-ROR1 antibody, and 2 represents the binding site with the drug; Most preferably, the anti-ROR1 antibody-drug conjugate has the following structure: Where n is 3 to 5.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the concentration of the anti-ROR1 antibody-drug conjugate is 2 mg / mL to 40 mg / mL, preferably 5 mg / mL to 15 mg / mL, and more preferably 10 mg / mL.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the buffer is a citrate buffer; preferably, the citrate buffer is a citrate-sodium citrate buffer or a citrate-sodium hydroxide buffer; more preferably, the concentration of the buffer is 10 mM to 50 mM, most preferably 15 mM to 30 mM; optionally, the concentration of the buffer is 20 mM.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the pH value of the pharmaceutical composition is 5.0 to 7.0; preferably, the pH value of the pharmaceutical composition is 5.0 to 6.5; more preferably, the pH value of the pharmaceutical composition is 5.0 to 6.

0.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the stabilizer is sucrose, trehalose, mannitol, or a combination thereof; preferably, The stabilizer is sucrose; more preferably, the concentration of the stabilizer is 60 mg / mL to 100 mg / mL; most preferably, the stabilizer is 80 mg / mL; or The stabilizer is a combination of sucrose and mannitol; more preferably, the stabilizer contains 5 mg / mL to 20 mg / mL of sucrose and 30 mg / mL to 60 mg / mL of mannitol; most preferably, the stabilizer contains 15 mg / mL of sucrose and 45 mg / mL of mannitol.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the surfactant is polysorbate; preferably, the polysorbate is polysorbate 20 or polysorbate 80; more preferably, the concentration of the surfactant is 0.01 mg / mL to 1.0 mg / mL, preferably 0.1 mg / mL to 0.5 mg / mL, and more preferably 0.2 mg / mL.

10. The pharmaceutical composition according to any one of claims 1 to 9, comprising: a) Anti-ROR1 antibody-drug conjugate at concentrations of 2 mg / mL to 40 mg / mL; b) 10mM~50mM citrate buffer; c) 60 mg / mL to 100 mg / mL sucrose or trehalose; d) 0.1 mg / mL to 0.5 mg / mL surfactant; the pH value of the pharmaceutical composition is 5.0 to 7.0; or a) Anti-ROR1 antibody-drug conjugate at concentrations of 2 mg / mL to 40 mg / mL; b) 10mM~50mM citrate buffer; c) 5 mg / mL to 20 mg / mL sucrose and 30 mg / mL to 60 mg / mL mannitol; d) 0.1 mg / mL to 0.5 mg / mL surfactant; the pH value of the pharmaceutical composition is 5.0 to 7.0; Preferably, the pharmaceutical composition comprises: a) 5 mg / mL to 15 mg / mL anti-ROR1 antibody-drug conjugate; b) 15mM~30mM citrate buffer; c) 70 mg / mL–90 mM mg / mL sucrose or trehalose; and d) 0.1 mg / mL to 0.5 mg / mL polysorbate 20 or polysorbate 80, wherein the pH of the pharmaceutical composition is 5.0 to 6.5; or The pharmaceutical composition comprises: a) 5 mg / mL to 15 mg / mL anti-ROR1 antibody-drug conjugate; b) 15mM~30mM citrate buffer; c) 5 mg / mL–20 mg / mL sucrose and 30 mg / mL–60 mg / mL mannitol; and d) 0.1 mg / mL to 0.5 mg / mL polysorbate 20 or polysorbate 80, wherein the pH of the pharmaceutical composition is 5.0 to 6.5; more preferably, The pharmaceutical composition comprises: a) 10 mg / mL anti-ROR1 antibody-drug conjugate; b) 20mM citrate-sodium citrate buffer; c) 80 mg / mL sucrose; and d) 0.2 mg / mL polysorbate 20, wherein the pH of the pharmaceutical composition is 5.0–6.5; or The pharmaceutical composition comprises: a) 10 mg / mL anti-ROR1 antibody-drug conjugate; b) 20mM citrate-sodium citrate buffer; c) 15 mg / mL sucrose and 45 mg / mL mannitol; and d) 0.2 mg / mL polysorbate 20, wherein the pH value of the pharmaceutical composition is 5.0–6.5; Optionally, the pharmaceutical composition comprises: a) 10 mg / mL anti-ROR1 antibody-drug conjugate; b) 20mM citrate-sodium citrate buffer; c) 80 mg / mL sucrose; and d) 0.2 mg / mL polysorbate 20, wherein the pH of the pharmaceutical composition is 5.

5.

11. A lyophilized formulation, said lyophilized formulation being prepared by freeze-drying the pharmaceutical composition of any one of claims 1 to 10.

12. The lyophilized formulation according to claim 11, wherein the lyophilization includes the steps of pre-freezing, primary drying, and secondary drying; preferably, the pre-freezing includes the following steps: a1. Cooling the pharmaceutical composition according to any one of claims 1 to 10 to a temperature of -40°C or lower; a2. Maintain isothermal temperature for at least 120 min; and / or The single freeze-drying process includes the following steps: b1. Warm the pharmaceutical composition from -40°C or lower to -20°C; b2. Maintain isothermal temperature for at least 25 hours.

13. The lyophilized formulation according to claim 12, wherein the vacuum degree of the primary drying and secondary drying is less than 0.12 mbar; preferably, the vacuum degree of the primary drying and secondary drying is 0.08 mbar.

14. A reconstituted solution, said reconstituted solution being prepared by reconstituted lyophilized formulation according to any one of claims 11 to 13; preferably, said reconstituted solution comprises: a) 5 mg / mL to 15 mg / mL anti-ROR1 antibody-drug conjugate; b) 15mM~30mM citrate buffer; c) 70 mg / mL to 90 mg / mL sucrose or trehalose; and d) 0.1 mg / mL to 0.5 mg / mL polysorbate 20 or polysorbate 80, wherein the pH of the pharmaceutical composition is 5.0 to 6.5; or a) 5 mg / mL to 15 mg / mL anti-ROR1 antibody-drug conjugate; b) 15mM~30mM citrate buffer; c) 5 mg / mL–20 mg / mL sucrose and 30 mg / mL–60 mg / mL mannitol; and d) 0.1 mg / mL to 0.5 mg / mL polysorbate 20 or polysorbate 80, wherein the pH of the pharmaceutical composition is 5.5 to 6.5; More preferably, the reconstituted solution comprises: a) 10 mg / mL anti-ROR1 antibody-drug conjugate; b) 20mM citrate-sodium citrate buffer; c) 80 mg / mL sucrose; and d) 0.2 mg / mL polysorbate 20, wherein the pH of the pharmaceutical composition is 5.

5.

15. An article comprising a container containing a pharmaceutical composition as claimed in any one of claims 1 to 10, a lyophilized formulation as claimed in any one of claims 11 to 13, or a reconstituted solution as claimed in claim 14.

16. Use of the pharmaceutical composition according to any one of claims 1 to 10, the lyophilized formulation according to any one of claims 11 to 13, or the reconstituted solution according to claim 14 in the preparation of a medicament for treating tumors or cancer; Preferably, the tumor or cancer is a solid tumor or a hematologic malignancy; more preferably, the tumor or cancer is selected from breast cancer, pancreatic cancer, lung cancer, esophageal cancer, laryngeal tumor, sarcoma, pharyngeal tumor, oral tumor, gastric cancer, ovarian cancer, prostate cancer, bladder cancer, colon cancer, colorectal cancer, urothelial carcinoma, neuroblastoma, cervical cancer, lymphoma, and leukemia; most preferably, the tumor or cancer expresses ROR1.

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