Pharmaceutical composition comprising antibody-drug conjugate targeting ROR1

Abstract

Provided in the present application are a pharmaceutical composition comprising an antibody-drug conjugate targeting ROR1 and use thereof in the preparation of a drug for treating or preventing cancers.

Description

Pharmaceutical compositions containing antibody-drug conjugates targeting ROR1 Technical Field

[0001] This application relates to the field of biopharmaceuticals, and more specifically, to a pharmaceutical composition comprising an antibody-drug conjugate targeting ROR1 and its use in the preparation of a medicament for the treatment or prevention of cancer, and a method thereof for the treatment or prevention of cancer. Background Technology

[0002] ROR1, short for Receptor-Tyrosine Kinase-Like Orphan Receptor 1, is a type I single-transmembrane protein and a member of the receptor tyrosine kinase (RTK) family. It is expressed at low levels during embryonic development but at high levels in various malignant tumors or tissues, such as chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), breast cancer, ovarian cancer, melanoma, and lung adenocarcinoma. Extensive data show that ROR1 plays a significant role in promoting tumor growth and metastasis, inducing drug resistance in tumor cells, and inhibiting apoptosis. The human ROR1 molecule consists of an extracellular region, a transmembrane region, and an intracellular region. The extracellular region includes an immunoglobulin-like domain (Ig-like), a cysteine-rich domain (CRD or FZD), and a Kring (KNG) domain (KRD). The intracellular region contains a tyrosine kinase domain (TKD), two serine / threonine-rich domains (Ser / ThrD), and a proline-rich domain (PRD).

[0003] In studies of antibody and antibody-drug conjugate (ADC) formulations, the formation of aggregates and the generation of breakdown products can cause pharmaceutically undesirable side effects, leading to increased immunogenicity or intravenous disease-related risks in patients receiving drug treatment. For these reasons, it is necessary to inhibit aggregate formation and breakdown product generation when formulating related formulations. Therefore, researchers have investigated various formulations and formulation methods for drug compositions (e.g., aqueous injections and lyophilized injections). Similarly, studying the formulation of antibody-drug conjugates presents further technical challenges, requiring consideration not only of the specific properties of the antibody moiety but also of the drug-connector moiety. For example, small molecule toxins may detach during storage of antibody-drug conjugates, which can affect the efficacy and toxicity of the drug.

[0004] Therefore, there remains a need in the art for stable antibody-drug conjugate formulations, especially formulations suitable for the antibody-drug conjugates of this application. Summary of the Invention

[0005] In view of the deficiencies of the prior art, this application provides a pharmaceutical composition comprising an antibody drug conjugate targeting ROR1, its use in the preparation of a drug for treating or preventing cancer, and a method thereof for treating or preventing cancer.

[0006] In a first aspect, this application provides a pharmaceutical composition comprising an antibody-drug conjugate targeting ROR1, comprising (i) an antibody-drug conjugate targeting ROR1, (ii) a buffer, (iii) a stabilizer, and (iv) a surfactant.

[0007] In some embodiments, the pharmaceutical composition comprises (i) 5.0–15.0 mg / mL of an antibody-drug conjugate targeting ROR1, (ii) 20.0–40.0 mM of a buffer, (iii) 5.0–10.0% (w / v) of a stabilizer, and (iv) 0.02–0.06% (w / v) of a surfactant. In a further embodiment, the pharmaceutical composition is a liquid formulation.

[0008] In some embodiments, the pharmaceutical composition comprises (i) 9.0–11.0 mg / mL of an antibody-drug conjugate targeting ROR1, (ii) 28.0–32.0 mM of a buffer, (iii) 7.0–9.0% (w / v) of a stabilizer, and (iv) 0.03–0.05% (w / v) of a surfactant. In a further embodiment, the pharmaceutical composition is a liquid formulation.

[0009] In some embodiments, the pharmaceutical composition comprises (i) 5.0% to 15.0% (wt%) of an antibody-drug conjugate targeting ROR1, (ii) 3.0% to 7.0% (wt%) of a buffer, (iii) 70.0% to 90.0% (wt%) of a stabilizer, and (iv) 0.01% to 1.00% (wt%) of a surfactant. In a further embodiment, the pharmaceutical composition is a solid dosage form; preferably, the solid dosage form is a lyophilized powder for injection, which is obtained by lyophilizing the liquid dosage form.

[0010] In some embodiments, the pharmaceutical composition comprises (i) 9.0% to 12.0% (wt%) of an antibody-drug conjugate targeting ROR1, (ii) 4.8% to 6.0% (wt%) of a buffer, (iii) 80.0% to 80.5% (wt%) of a stabilizer, and (iv) 0.30% to 0.50% (wt%) of a surfactant. In a further embodiment, the pharmaceutical composition is a solid dosage form; preferably, the solid dosage form is a lyophilized powder for injection, which is obtained by lyophilizing the liquid dosage form.

[0011] In some embodiments, the antibody-drug conjugate targeting ROR1 has a structure represented by Formula I: A-(LD) d (Formula I), where A is an antibody or its antigen-binding fragment targeting ROR1; D is the drug portion, selected from microtubule inhibitors of MMAE or its analogues; L is the linker portion, connected to A at one end and to the drug portion D at the other end; d is an integer from 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12) or a decimal.

[0012] In some embodiments, the antibody targeting ROR1 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, each of which contains three CDRs. The heavy chain variable region comprises: CDR1-H, having the amino acid sequence shown in SEQ ID NO:1; CDR2-H, having the amino acid sequence selected from SEQ ID NO:2, 9, and 11; and CDR3-H, having the amino acid sequence shown in SEQ ID NO:3. The light chain variable region comprises: CDR1-L, having the amino acid sequence shown in SEQ ID NO:4; CDR2-L, having the amino acid sequence shown in SEQ ID NO:5; and CDR3-L, having the amino acid sequence shown in SEQ ID NO:6, encoded using Kabat encoding.

[0013] In some embodiments, the antibody targeting ROR1 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, each of which contains three CDRs. The heavy chain variable region comprises: CDR1-H, having the amino acid sequence shown in SEQ ID NO:1; CDR2-H, having the amino acid sequence shown in SEQ ID NO:2; and CDR3-H, having the amino acid sequence shown in SEQ ID NO:3. The light chain variable region comprises: CDR1-L, having the amino acid sequence shown in SEQ ID NO:4; CDR2-L, having the amino acid sequence shown in SEQ ID NO:5; and CDR3-L, having the amino acid sequence shown in SEQ ID NO:6, using Kabat encoding.

[0014] In some embodiments, the antibody targeting ROR1 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, each of which contains three CDRs. The heavy chain variable region comprises: CDR1-H, having the amino acid sequence shown in SEQ ID NO:1; CDR2-H, having the amino acid sequence shown in SEQ ID NO:9; and CDR3-H, having the amino acid sequence shown in SEQ ID NO:3. The light chain variable region comprises: CDR1-L, having the amino acid sequence shown in SEQ ID NO:4; CDR2-L, having the amino acid sequence shown in SEQ ID NO:5; and CDR3-L, having the amino acid sequence shown in SEQ ID NO:6, using Kabat encoding.

[0015] In some embodiments, the antibody targeting ROR1 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, each of which contains three CDRs. The heavy chain variable region comprises: CDR1-H, having the amino acid sequence shown in SEQ ID NO:1; CDR2-H, having the amino acid sequence shown in SEQ ID NO:11; and CDR3-H, having the amino acid sequence shown in SEQ ID NO:3. The light chain variable region comprises: CDR1-L, having the amino acid sequence shown in SEQ ID NO:4; CDR2-L, having the amino acid sequence shown in SEQ ID NO:5; and CDR3-L, having the amino acid sequence shown in SEQ ID NO:6, using Kabat encoding.

[0016] In some embodiments, the antibody targeting ROR1 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, each of which contains three CDRs. The heavy chain variable region comprises: CDR1-H, having the amino acid sequence shown in SEQ ID NO:20; CDR2-H, having the amino acid sequence shown in SEQ ID NO:21; and CDR3-H, having the amino acid sequence shown in SEQ ID NO:22. The light chain variable region comprises: CDR1-L, having the amino acid sequence shown in SEQ ID NO:23; CDR2-L, having the amino acid sequence of AAT; and CDR3-L, having the amino acid sequence shown in SEQ ID NO:6, encoded using IMGT.

[0017] In some embodiments, the antibody targeting ROR1 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, each of which contains three CDRs. The heavy chain variable region comprises: CDR1-H, having the amino acid sequence shown in SEQ ID NO:24; CDR2-H, having the amino acid sequence shown in SEQ ID NO:25; and CDR3-H, having the amino acid sequence shown in SEQ ID NO:3. The light chain variable region comprises: CDR1-L, having the amino acid sequence shown in SEQ ID NO:4; CDR2-L, having the amino acid sequence shown in SEQ ID NO:5; and CDR3-L, having the amino acid sequence shown in SEQ ID NO:6, encoded in a Chothia manner.

[0018] In some embodiments, the antibody targeting ROR1 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, each containing three CDRs. The heavy chain variable region comprises: CDR1-H, having the amino acid sequence shown in SEQ ID NO:26; CDR2-H, having the amino acid sequence shown in SEQ ID NO:27; and CDR3-H, having the amino acid sequence shown in SEQ ID NO:28. The light chain variable region comprises: CDR1-L, having the amino acid sequence shown in SEQ ID NO:29; CDR2-L, having the amino acid sequence shown in SEQ ID NO:30; and CDR3-L, having the amino acid sequence shown in SEQ ID NO:31, using a Contact encoding scheme. In some embodiments, the antibody A targeting ROR1 is selected from antibodies Hu17-H2L1, Hu17-H3L1, and Hu17-H4L1.

[0019] In some embodiments, the buffer is selected from histidine buffers, citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, and phosphate buffers. In some embodiments, the buffer is a histidine buffer; preferably, the histidine buffer is histidine-histidine hydrochloride.

[0020] In some embodiments, the pharmaceutical composition is a liquid formulation (e.g., an aqueous injection) with a pH of 6.0 to 6.6; or a solid formulation (e.g., a lyophilized powder for injection) obtained by lyophilizing the liquid formulation (e.g., an aqueous injection).

[0021] In some embodiments, the stabilizer is selected from sucrose, trehalose, mannitol, sorbitol, L-serine, monosodium glutamate, alanine, glycine, sarcosine, proline, and methionine. In some embodiments, the stabilizer is trehalose.

[0022] In some embodiments, the surfactant is polysorbate or poloxamer; preferably, the surfactant is selected from polysorbate 80, polysorbate 20, and poloxamer 188. In some embodiments, the surfactant is polysorbate 20.

[0023] In some embodiments, (a) the buffer is selected from histidine buffers, citrate buffers, and succinate buffers; preferably, the buffer is a histidine buffer; more preferably, the histidine buffer is histidine-histidine hydrochloride; (b) the stabilizer is selected from sucrose and trehalose; preferably, the stabilizer is trehalose; (c) the surfactant is selected from polysorbate 80 and polysorbate 20; preferably, the surfactant is polysorbate 20. In some embodiments, (a) the buffer is histidine-histidine hydrochloride; (b) the stabilizer is trehalose; and (c) the surfactant is polysorbate 20.

[0024] In some embodiments, the pharmaceutical composition is prepared in a form selected from solid dosage forms, semi-solid dosage forms, liquid dosage forms, and gaseous dosage forms; preferably, the pharmaceutical composition is prepared in the form of a liquid dosage form or a solid dosage form; more preferably, the pharmaceutical composition is prepared in the form of a solid dosage form (e.g., a lyophilized powder for injection). In some embodiments, the lyophilized powder for injection is obtained by lyophilizing the liquid dosage form.

[0025] In some embodiments, the pharmaceutical composition is prepared as a liquid formulation containing: 5.0–15.0 mg / mL of an antibody-drug conjugate targeting ROR1, 20.0–40.0 mM of histidine-histidine hydrochloride, 5.0–10.0% (w / v) trehalose, 0.02–0.06% (w / v) polysorbate 20, and pH 6.0–6.6.

[0026] In some embodiments, the pharmaceutical composition is prepared as a liquid formulation containing: about 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, or 15.0 mg / mL of an antibody-drug conjugate targeting ROR1; about 20.0, 25.0, 30.0, 35.0, or 40.0 mM of histidine-histidine hydrochloride; about 5.0%, 5.5%, ... The liquid formulation contains 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, or 10.0% (w / v) trehalose; and about 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, 0.05%, 0.055%, or 0.06% (w / v) polysorbate 20, with a pH of about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, or 6.6. In a further embodiment, the liquid formulation contains water; preferably, the water is water for injection.

[0027] In some embodiments, the pharmaceutical composition is prepared as a liquid formulation containing: 9.0–11.0 mg / mL of an antibody-drug conjugate targeting ROR1, 28.0–32.0 mM of histidine-histidine hydrochloride, 7.0–9.0% (w / v) trehalose, 0.03–0.05% (w / v) polysorbate 20, and pH 6.0–6.6.

[0028] In some embodiments, the pharmaceutical composition is prepared as a liquid formulation containing: about 10.0 mg / mL of an antibody-drug conjugate targeting ROR1, about 30.0 mM of histidine-histidine hydrochloride, about 8.0% (w / v) of trehalose, about 0.04% (w / v) of polysorbate 20, and a pH of about 6.3.

[0029] In some embodiments, the pharmaceutical composition is prepared as a liquid formulation containing: 10.0 mg / mL of an antibody-drug conjugate targeting ROR1, 30.0 mM histidine-histidine hydrochloride, 8.0% (w / v) trehalose, 0.04% (w / v) polysorbate 20, and a pH of 6.3. In a further embodiment, the liquid formulation contains water; preferably, the water is water for injection.

[0030] In some embodiments, the liquid formulation further contains water; preferably, the water is water for injection.

[0031] In some embodiments, the pharmaceutical composition is prepared as a solid dosage form (e.g., a lyophilized powder for injection) containing: 5.0% to 15.0% (wt%) of an antibody-drug conjugate targeting ROR1, 2.0% to 4.0% (wt%) of histidine, 1.0% to 3.0% (wt%) of histidine hydrochloride, 70.0% to 90.0% (wt%) of trehalose, and 0.01% to 1.00% (wt%) of polysorbate 20.

[0032] In some embodiments, the pharmaceutical composition is prepared as a solid dosage form (e.g., a lyophilized powder for injection) containing: about 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 10.5%, 11.0%, 11.5%, 12.0%, 12.5%, 13.0%, 13.5%, 14.0%, 14.5%, or 15.0% (wt%) of an antibody-drug conjugate targeting ROR1; and about 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3. 6%, 3.7%, 3.8%, 3.9%, or 4.0% (wt%) of histidine; approximately 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, or 3% (wt%) of histidine. 0.0% (wt%) of histidine hydrochloride; about 70.0%, 75.0%, 80.0%, 85.0% or 90.0% (wt%) of trehalose; and about 0.01%, 0.10%, 0.20%, 0.30%, 0.40%, 0.50%, 0.60%, 0.70%, 0.80%, 0.90% or 1.00% (wt%) of polysorbate 20.

[0033] In some embodiments, the pharmaceutical composition is prepared as a solid dosage form (e.g., a lyophilized powder for injection) containing: 9.0% to 12.0% (wt%) of an antibody-drug conjugate targeting ROR1, 2.8% to 3.4% (wt%) of histidine, 2.0% to 2.5% (wt%) of histidine hydrochloride, 80.0% to 80.5% (wt%) of trehalose, and 0.30% to 0.50% (wt%) of polysorbate 20.

[0034] In some embodiments, the pharmaceutical composition is prepared as a solid dosage form (e.g., a lyophilized powder for injection) containing: about 10.46% (wt%) of an antibody-drug conjugate targeting ROR1, about 3.24% (wt%) of histidine, about 2.20% (wt%) of histidine hydrochloride, about 83.68% (wt%) of trehalose, and about 0.42% (wt%) of polysorbate 20.

[0035] In some embodiments, the pharmaceutical composition is prepared in the form of a solid dosage form (e.g., a lyophilized powder for injection) to remove moisture as much as possible.

[0036] In some embodiments, the lyophilized powder injection is prepared through a pre-freezing step, a primary drying step, and a desorption drying step. In some embodiments, the lyophilized powder injection is prepared from the aforementioned liquid formulation through a pre-freezing step, a primary drying step, and a desorption drying step.

[0037] In some embodiments, the pre-freezing step is performed at a temperature of -40°C, using a cooling rate of 1–2°C / min (preferably 2°C / min). In a further embodiment, the pre-freezing step further includes maintaining the plate at 4°C for 10–60 min (preferably 30 min) before the pre-freezing cooling begins.

[0038] In some implementations, the primary drying step is carried out at a high temperature and low pressure of -5°C and a vacuum of 0.08 mbar.

[0039] In some implementations, the analytical drying step is carried out at 35°C and a vacuum of 0.20 mbar.

[0040] In some implementations, the antigen-binding fragment targeting ROR1 is a Fab fragment, an F(ab')2 fragment, or a single-chain Fv fragment (scFv).

[0041] In some embodiments, the amino acid sequence of the heavy chain variable region is selected from the amino acid sequences shown in SEQ ID NO. 7, SEQ ID NO. 10, and SEQ ID NO. 12, and the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO. 8. In some embodiments, the amino acid sequence of the heavy chain variable region is selected from the amino acid sequence shown in SEQ ID NO. 10, and the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO. 8.

[0042] In some embodiments, the heavy chain of the antibody comprises a heavy chain constant region of the amino acid sequence shown in SEQ ID NO. 13, and the light chain of the antibody comprises a light chain constant region of the amino acid sequence shown in SEQ ID NO. 19.

[0043] In some embodiments, the C-terminus of the light chain of the antibody or its antigen-binding fragment is attached to a glutamine-containing tag peptide selected from LQSGA, GGLQSGA, and GGGLQSGA.

[0044] In some embodiments, the heavy chain of the antibody comprises (or is) an amino acid sequence region as shown in SEQ ID NO. 13, and the light chain of the antibody comprises (or is) an amino acid sequence as shown in SEQ ID No. 14.

[0045] In some embodiments, the microtubule inhibitor of the pharmaceutical fraction MMAE or its analogues is selected from auristatin E (AE), auristatin F (AF), monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), monomethylauristatin D (MMAD), dolastatin, and auristatin E 5-benzoylvalerate (AEVB). In some embodiments, the microtubule inhibitor of the pharmaceutical fraction MMAE or its analogues is monomethylauristatin E (MMAE).

[0046] In some embodiments, the L-linker portion is independently selected from -mc-Val-Cit-pAB-, -mc-Val-Cit-pABC-, -mc-Val-Cit-, -NH-(CH2-CH2-O)m-Val-Cit--NH-(CH2-CH2-O)m-Val-Cit-pABC-, and -NH-(CH2-CH2-O)m-CH2-C(=O)-Val-Cit-pABC-, wherein m in -(CH2-CH2-O)m- is an integer from 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8); preferably m is 3. In some embodiments, the linker portion is -NH-(CH2-CH2-O)3-CH2-C(=O)-Val-Cit-pABC-.

[0047] In some embodiments, the antibody-drug conjugate targeting ROR1 has a structure represented by Formula II:

[0048] Formula II, where A and d are defined as described above. In a further embodiment, A is selected from antibodies Hu17-H2L1, Hu17-H3L1, and Hu17-H4L1, and d is selected from integers or decimals from 1 to 8.

[0049] In some embodiments, the antibody-drug conjugate targeting ROR1 is selected from Hu17-H2L1-4LND1002, Hu17-H3L1-4LND1002, and Hu17-H4L1-4LND1002.

[0050] Hu17-H2L1-4LND1002:

[0051] Hu17-H3L1-4LND1002:

[0052] Hu17-H4L1-4LND1002:

[0053] In some embodiments, the antibody contains Q295 in its heavy chain constant region, and the linker portion is linked to the side chain of Q295 via an amide bond, and / or the linker portion is linked to the side chain of a glutamine residue in the glutamine-containing tag peptide GGLQSGA via an amide bond. In some embodiments, the antibody contains Q295 in its heavy chain constant region, and the linker portion is linked to the side chain of Q295 via an amide bond, and the linker portion is linked to the side chain of a glutamine residue in the glutamine-containing tag peptide GGLQSGA via an amide bond.

[0054] The various implementation schemes in the first aspect above can be combined in any way to form other implementation schemes, as long as they do not contradict each other.

[0055] In a second aspect, this application provides a method for treating or preventing cancer in an individual, comprising administering a therapeutically effective amount of the pharmaceutical composition described in the first aspect to an individual suffering from said cancer.

[0056] Thirdly, this application provides the use of the pharmaceutical composition described in the first aspect in the preparation of a medicament for treating or preventing cancer.

[0057] In the second and third aspects:

[0058] In some embodiments, the cancer is a solid tumor, such as gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, or gallbladder cancer, preferably adenocarcinoma of the stomach, esophagus, pancreatic duct, bile duct, lung, or ovary; more preferably gastric cancer or pancreatic cancer.

[0059] In some embodiments, the cancer is a hematologic malignancy, preferably lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, non-Hodgkin's lymphoma, Richter-transformed non-Hodgkin's lymphoma, T-cell leukemia, Burkitt's lymphoma, multiple myeloma, marginal zone lymphoma, small lymphocytic lymphoma, marginal zone lymphoma, or marginal cell B-cell lymphoma.

[0060] Fourthly, this application provides a method for preparing a lyophilized powder injection form of the pharmaceutical composition described in the first aspect, the method comprising a pre-freezing step, a primary drying step, and a desorption drying step of the pharmaceutical composition in liquid form.

[0061] In some embodiments, the pre-freezing step is performed at a temperature of -40°C, using a cooling rate of 1–2°C / min (preferably 2°C / min). In a further embodiment, the pre-freezing step further includes maintaining the plate at 4°C for 10–60 min (preferably 30 min) before the pre-freezing cooling begins. In a further embodiment, the pre-freezing time is preferably 150 min–200 min (more preferably about 180 min).

[0062] In some embodiments, the primary drying step is performed at a high temperature and low pressure of -5°C and a vacuum of 0.08 mbar. In a further embodiment, the primary drying time is preferably 2000 min to 2500 min (more preferably about 2200 min).

[0063] In some embodiments, the desorption drying step is performed at 35°C and a vacuum of 0.20 mbar. In a further embodiment, the desorption drying time is preferably 800 min to 1000 min (more preferably about 900 min). Attached image description:

[0064] Figure 1: Schematic diagram of the structure of the conjugate Hu17-H2L1 / H3L1 / H4L1-4LND1002ADC according to an exemplary embodiment of this application.

[0065] The left side of the figure shows the structure of the antibody, and the right side shows the corresponding linker-drug structure. Each molecule consists of one anti-ROR1 humanized monoclonal antibody (Hu17-H2L1 / H3L1 / H4L1) with one MMAE molecule linked to the Q295 (EU numbering) amino acid of each heavy chain via a linker. At the same time, a glutamine-containing tag peptide (GGLQSGA) is also linked to the C-terminus (carboxyl terminus) of each light chain, which is also linked to one MMAE molecule via a linker. The antibody and linker are linked by a stable amide bond (isopeptide bond), and the average drug-to-antibody ratio (DAR) is 4.0.

[0066] The right side of the figure shows the structure of LND1002 before conjugation, which includes a drug molecule, such as MMAE or an MMAE derivative, and a linker. After conjugation, the terminal NH2 group of the linker is linked to the side chain (-CH2-CH2-C(=O)-NH2) of the glutamine residue (Q) in the glutamine-containing tag peptide at the end of the heavy chain Q295 or the light chain to form the structure "-NH-(CH2-CH2-O)3-CH2-C(=O)-Val-Cit-pABC".

[0067] Figure 2: Affinity of murine and humanized antibodies to MDA-MB-231-ROR1 at the cellular level.

[0068] Figure 3: UC961-VC MMAE DAR detection spectrum.

[0069] Figure 4: Inhibitory curves of each ADC on human lung adenocarcinoma H1975 mouse xenografts.

[0070] Figure 5: Inhibitory curves of each ADC on human breast cancer HCC1187 mouse xenografts.

[0071] Figure 6: Phenomorphic photographs of freeze-dried powder containing 8% sucrose stabilizer and 8% trehalose stabilizer in batch A of sample Hu17-H2L1-4LND1002.

[0072] Figure 7: Photographs of the appearance of freeze-dried powder prepared by two primary drying parameters. Top: High temperature and low pressure -5℃, vacuum degree 0.08mbar; Bottom: Low temperature and high pressure -20℃, vacuum degree 0.2mbar. Detailed implementation method:

[0073] The embodiments listed below are provided to better illustrate the content of this application, but are not intended to limit the scope of this application to the illustrated embodiments. Non-essential improvements and adjustments made to the implementation schemes by those skilled in the art based on the above-described invention are still within the protection scope of this application.

[0074] Terminology Explanation:

[0075] Unless otherwise stated, as used herein, the singular forms “a,” “an,” and “the” also include the plural forms. For example, the term “a cell” includes multiple cells and mixtures thereof.

[0076] As used herein, the term "about" refers to the typical range of error for various values ​​that is readily known to those skilled in the art. The use of the value or parameter of "about" herein includes (and describes) embodiments involving that value or parameter itself.

[0077] Although the numerical ranges and parameter approximations shown in the broad scope of this application are intended to be as accurate as possible in the specific embodiments, any numerical value inherently contains a certain degree of error due to the standard deviation present in their respective measurements. Furthermore, all ranges disclosed herein should be understood to encompass any and all subranges contained therein. For example, the ranges described as “1 to 10” or “1 to 10” should be considered to include any and all subranges between the minimum value of 1 and the maximum value of 10 (inclusive); that is, all subranges starting with a minimum value of 1 or greater, such as 1 to 6.1, and subranges ending with a maximum value of 10 or less, such as 5.5 to 10.

[0078] As used herein, the terms “comprising,” “including,” or “containing” mean to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this application, when the terms “comprising,” “including,” or “containing” are used, they also cover the situation where the elements, integers, or steps mentioned are constituted, unless otherwise specified. For example, when referring to an antibody variable region that “comprising,” “including,” or “containing” a specific sequence, it is also intended to cover the antibody variable region composed of that specific sequence.

[0079] As used herein, the term "cancer" refers to a proliferative disorder caused by or characterized by cell proliferation, in which cells have lost their susceptibility to normal growth control. The term "cancer" includes tumors and any other proliferative disorder. Cancers of the same tissue type originate in the same tissue and can be classified into different subtypes based on their biological characteristics.

[0080] As used herein, the term "treatment" refers to a clinical intervention aimed at altering the natural course of the disease in the individual or cells receiving the treatment during the clinicopathological process. Ideal outcomes of treatment include slowing or reducing the rate of disease progression, improving or alleviating the disease state, and mitigating or improving prognosis. For example, if one or more symptoms associated with cancer are relieved or eliminated, including but not limited to reducing the proliferation of cancer cells (or eliminating cancer cells), reducing symptoms caused by the disease, improving the patient's quality of life, reducing the dosage of other medications required to treat the disease, slowing disease progression, and / or prolonging the patient's survival, then the patient has been successfully "treated."

[0081] The term “prevention” includes: (1) suppressing the onset of disease in a subject or patient who may be at risk and / or susceptible to the disease but has not yet experienced or exhibited any or all symptoms of the disease; and / or (2) slowing the onset of symptoms of the disease in a subject or patient who may be at risk and / or susceptible to the disease but has not yet experienced or exhibited any or all symptoms of the disease.

[0082] For specific details, professionals can refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in this field to refer to one of the 20 commonly used L-amino acids.

[0083] As used herein, the term "full-length antibody" refers to a complete or substantially complete antibody, as opposed to an antibody fragment. Specifically, full-length antibodies include those containing both a heavy chain and a light chain, including an Fc region. The constant domain can be a native sequence constant domain (e.g., a human native sequence constant domain) or a variant of its amino acid sequence. In some cases, a complete antibody possesses one or more effector functions.

[0084] Complementarity-determining regions (CDRs, typically CDR1, CDR2, and CDR3) are the regions in the variable region that have the greatest impact on antibody affinity and specificity. There are several common definitions for the CDR sequence of VH or VL, including Kabat, IMGT, Chothia, and Contact definitions. For a given antibody's variable region sequence, the CDR sequence in the VH and VL sequences can be determined according to the Kabat, IMGT, Chothia, or Contact definitions. In this application, unless otherwise expressly stated, the amino acid sequences of the CDRs described herein (e.g., SEQ ID Nos. 1-6, 9, 11) are all as shown in the Kabat definition. However, it is well known to those skilled in the art that antibody CDRs can be defined in various ways. Although the scope of protection claimed in this application is based on the sequence shown in the Kabat definition, the amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of this application.

[0085] In this application, unless otherwise expressly stated, the amino acid sequences of the CDRs described herein (e.g., SEQ ID No. 1-6, 9, 11) are as shown in accordance with the Kabat definition rules.

[0086] As used in this article, the antibody-related term "heavy chain variable region (VH)" refers to the amino-terminal variable region domain of the immunoglobulin heavy chain.

[0087] As used in this article, the antibody-related term "light chain variable region (VL)" refers to the amino-terminal variable region domain of the immunoglobulin light chain.

[0088] As used herein, the term "antigen-binding fragment" refers to an antibody fragment such as Fv, Fab, F(ab')2, or Fab', or any fragment that should be able to increase its half-life through chemical modification or incorporation into liposomes, such as the addition of a poly(alkylene) glycol such as polyethylene glycol ("PEGylated, PEGylated") (a PEGylated fragment referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG, or Fab'-PEG) ("PEG" stands for polyethylene glycol), which has ROR1 binding activity. Preferably, the antigen-binding fragment will consist of or contain a portion of the variable region of the heavy or light chain of its source antibody, the portion of which is sufficient to retain the same binding specificity and sufficient affinity as its source antibody, and such functional fragment will contain at least 5 amino acids, preferably 10, 15, 25, 50, and 100 consecutive amino acids of its source antibody sequence. Examples of antigen-binding fragments include, but are not limited to: (1) Fab fragments, which may be monovalent fragments having VL-CL chains and VH-CH1 chains; (2) F(ab')2 fragments, which may be divalent fragments having two Fab' fragments connected by disulfide bridges (i.e., Fab' dimers) in the hinge region; and (3) Fv fragments with VL and VH domains on a single arm of antibody.

[0089] The term "therapeutic effective dose" or "effective dose" as used in this article refers to a dose sufficient to demonstrate its benefit to the recipient. The actual amount administered, as well as the rate and duration of administration, will depend on the individual's condition and the severity of the illness.

[0090] The term "object" as used in this article refers to mammals, such as humans, but can also be other animals, such as wild animals (e.g., herons, storks, cranes, etc.), livestock (e.g., ducks, geese, etc.) or laboratory animals (e.g., chimpanzees, monkeys, rats, mice, rabbits, guinea pigs, marmots, ground squirrels, etc.).

[0091] The antibody-drug conjugate (ADC) described in this application refers to a drug with cytotoxicity conjugated to an antibody, wherein the antigen of the antibody is expressed on the surface of cancer cells and the antibody also binds to an antigen that can be internalized into cells, and thus can selectively deliver the drug to cancer cells, thereby causing the drug to accumulate in the cancer cells and kill the cancer cells.

[0092] As used herein, the term "buffer" refers to a pharmaceutically acceptable buffer. The term "buffer" encompasses substances that maintain the pH of a solution within, for example, an acceptable range, including but not limited to histidine buffers, citrate buffers, and succinate buffers as described herein.

[0093] As used herein, "histidine (salt) buffer" and "histidine (salt) buffer" are buffers containing histidine ions. Examples of histidine buffers include histidine-histidine hydrochloride, histidine-acetate, histidine-phosphate, and histidine-sulfate buffers. Histidine-histidine hydrochloride buffers are prepared by reacting histidine with histidine hydrochloride, or by reacting histidine with hydrochloric acid.

[0094] As used herein, “citrate buffer” or “citrate buffer solution” refers to a buffer containing citrate ions. Examples of citrate buffers include sodium citrate, potassium citrate, calcium citrate, magnesium citrate, etc.

[0095] As used herein, "succinate buffer" or "succinate buffer solution" refers to a buffer containing succinate ions. Examples of succinate buffers include sodium succinate-succinate, potassium succinate-succinate, calcium succinate-succinate, etc. For example, the sodium succinate-succinate may be prepared from succinic acid and sodium hydroxide, or from succinic acid and sodium succinate.

[0096] As used herein, the term "stabilizer" includes substances that provide stability to proteins, such as those used as cryoprotectants during freezing and / or as lyophilization protectants during (freeze-)drying or "dehydration" processes.

[0097] As used herein, the term "surfactant" is typically used in pharmaceutical formulations to prevent drug adsorption to surfaces and / or aggregation. Furthermore, surfactants reduce surface tension (or interfacial tension) between two liquids or between a liquid and a solid.

[0098] As used herein, the term "w / v" refers to a weight / volume percentage concentration, used to describe the concentration of solute in a solution. For example, 4% (w / v) means that there are 4 grams of solute per 100 ml of solution.

[0099] The term "wt%" indicates a percentage by mass.

[0100] The term "UC961-vc-MMAE," also known as VLS-101, is an ADC (antibody-adjuvant antibody) obtained by conjugating the microtubule polymerization inhibitor MMAE to the cysteine ​​residue of the antibody Cirmtuzumab (UC961) using the cleavable linker MC-Val-Cit-PAB. It has a mean DAR of 4. The antibody sequence can be found in sequences 3-6 of CN111587124A.

[0101] Example

[0102] To facilitate understanding of this application, reference will be made to certain embodiments, and specific language will be used to describe the application. However, it should be understood that these specific embodiments are not intended to limit the scope of this application. Any changes and further modifications to the described embodiments, as well as any further direct or indirect application of this application, are those that would normally occur to those skilled in the art.

[0103] Example 1: Construction of humanized expression vector and preparation of plasmids

[0104] The murine antibody 17-F12-G8-A6 was humanized to obtain three humanized antibodies: hu17-H2L1, hu17-H3L1, and hu17-H4L1. The amino acid sequences of the variable regions of the heavy and light chains of 17-F12-G8-A6 are shown in SEQ ID No. 17 and SEQ ID No. 18 of the sequence listing, respectively; the amino acid sequences of the variable regions of the heavy and light chains of hu17-H3L1 are shown in SEQ ID No. 7 and SEQ ID No. 8 of the amino acid sequence listing, respectively; the amino acid sequences of the variable regions of the heavy and light chains of hu17-H2L1 are shown in SEQ ID No. 10 and SEQ ID No. 8 of the amino acid sequence listing, respectively; the amino acid sequences of the variable regions of the heavy and light chains of hu17-H4L1 are shown in SEQ ID No. 12 and SEQ ID No. 8 of the amino acid sequence listing, respectively; the amino acid sequences of the constant regions of the heavy and light chains of hu17-H2L1, hu17-H3L1, and hu17-H4L1 are shown in SEQ ID No. 13 and SEQ ID No. 14 of the amino acid sequence listing, respectively.

[0105] Plasmid preparation:

[0106] The target sequence was cloned by PCR, and the PCR product was purified by gel electrophoresis. The target fragment was obtained using a gel extraction kit (Tiangen). The gel extraction product and plasmid vector PCDNA3.4 were ligated and transformed to obtain a recombinant plasmid. The plasmid was transformed into E. coli and plated on plates containing ampicillin resistance for colony screening. Random colonies were selected for sequencing. Positive colonies with correct sequencing were expanded and cultured, and the plasmid was extracted using an endotoxin-free plasmid extraction kit (Tiangen) for further sequencing verification.

[0107] Example 2: Expression of humanized monoclonal antibodies

[0108] 1) Take 145 × 10⁻⁶ CHO cells 6 One, centrifuge to remove the supernatant.

[0109] 2) Add about 0.5 mL of electroporation buffer to the cells, mix well, and then add an appropriate amount of plasmid (concentration 500 ng / uL).

[0110] 3) After thoroughly mixing the above cell and plasmid suspensions, take 1 mL, add it to the electroporation tube, and place the electroporation tube into the electroporation apparatus for electroporation.

[0111] 4) After electroporation, aliquot the cells from the electroporation tube into shake flasks containing 20 mL of culture medium and incubate statically for 40 min.

[0112] 5) After incubation, place the shake flasks in a 37°C, 270 rpm, 8% CO2 incubator. After 24 hours, add feed / sodium butyrate / double antibody and continue incubation for 3-7 days. On day 5, take samples for ELISA testing to confirm the correct expression of humanized monoclonal antibodies hu17-H2L1, hu17-H3L1, and hu17-H4L1.

[0113] Example 3: Monoclonal Antibody Purification

[0114] 1) Using the AKTA purification system, first equilibrate the chromatography column: 1xPBS, flow rate 1mL / min, 20mL;

[0115] 2) Sample loading: Flow rate 1 mL / min;

[0116] 3) Washing: 1xPBS, flow rate 1mL / min, 20mL;

[0117] 4) Elution: Sodium acetate buffer (pH 3.4), 1 mL / min, collected in aliquots, approximately 500 μL per tube. A total of 10 tubes were collected, and the absorbance at 280 nm was read using a NanoDrop instrument.

[0118] 5) Dialysis: Aspirate the high concentration of protein into a dialysis bag and place it into a beaker containing 1x PBS pH 7.0 for dialysis.

[0119] 6) Obtain monoclonal antibodies with an antibody purity (SEC-HPLC) > 95%.

[0120] Example 4: Affinity assessment of humanized monoclonal antibodies

[0121] In this experiment, FACS was used to assess the affinity of the humanized ROR1 antibody for the stable transgenic cell line MDA-MB-231-ROR1 (Kangyuan Bochuang) expressing ROR1. ROR1 antigen-positive cells MDA-MB-231-ROR1 were digested with trypsin and collected, with 1 × 10⁶ cells per well. 5 Cells were seeded into 96-well plates, and serially diluted antibodies (starting at 15 μg, 5-fold dilution, 11 concentration points) were added. After incubation at 37°C for 2 h, the cells were washed with PBS, and secondary antibody binding to IgG (goat anti-human cross-adsorption secondary antibody) was added. The cells were incubated at 37°C for 1 h. The absorbance was detected by flow cytometry, and the curve was fitted using GraphPad software. The experimental results are shown in Table 1 and Figure 2.

[0122] Table 1. Results of FACS affinity assay for murine and humanized antibodies.

[0123] The results showed that, after humanization, the affinity of the anti-ROR1 humanized antibodies hu17-H2L1, hu17-H3L1 and hu17-H4L1 for the ROR1-expressing tumor cells MDA-MB-231-ROR1 was comparable to that of the murine antibody 17-F12-G8-A6.

[0124] Example 5: ADC Preparation

[0125] 1. UC961-vcMMAE

[0126] The UC961 antibody was displaced into a PBS 7.0 / 5mM EDTA buffer to a concentration of 10 mg / mL. Three molar equivalents of TCEP reducing agent were added according to the antibody's molar concentration, and the mixture was heated in a 37°C water bath for 1 h. Then, 2.5 molar equivalents of VC MMAE (CAS No: 646502-53-6) were added, and the reaction was carried out at room temperature for 30 min. Finally, 2.5 molar equivalents of N-acetyl-L-cysteine ​​were added, and the reaction was stopped after 30 min. After stopping the reaction, free small molecules and other impurities were removed by ultrafiltration concentration and buffer replacement to obtain UC961-VC MMAE.

[0127] 2. Preparation of samples Hu17-H2L1-4LND1002, Hu17-H3L1-4LND1002, and Hu17-H4L1--4LND1002

[0128] A certain amount of LND1002, Hu17-H2L1, Hu17-H3L1 or Hu17-H4L1 antibody, mTGase (amino acid sequence SEQ ID NO.15, the enzyme used in all the following examples), and H2O were added to an EP tube, sealed and mixed, and placed at room temperature for no more than 4 days. mTGase catalyzes the formation of stable amide bonds (isopeptide bonds) between LND1002 and the antibody glutamine amino acids. When the heavy chain coupling rate exceeds 95%, the conjugation reaction is considered complete, and purification is performed immediately.

[0129] Using a sterilized AKTA system, the conjugated reactants were loaded onto a sterilized Protein A (MabSelect Sure) column at 30-35 g ADC / L resin, with a minimum residence time of 5 minutes to ensure complete binding of the ADC product. The column was then washed with excess binding and washing buffer (1×PBS pH 7.0) to remove mTGase, unreacted LND1002, and any unwanted buffer components before the desired product was eluted at a low pH. The eluted fraction was collected in collection tubes pre-filled with neutralization buffer to obtain antibody-drug conjugates Hu17-H2L1-4LND1002, Hu17-H3L1-4LND1002, and Hu17-H4L1-4LND1002.

[0130] Example 6: Determination of coupling sites in Hu17-H2L1-4LND1002, Hu17-H3L1-4LND1002, and Hu17-H4L1-4LND1002 samples

[0131] ADC samples were digested using protease. The digested peptides were detected using ultra-high performance liquid chromatography (UHPLC) and high-resolution mass spectrometry (HPLC-MS / MS). The raw HPLC data were analyzed using UNIFI software in UPLC-MS / MS to identify modification sites and their intensity. 100 μg of ADC sample was added to a final concentration of 6 M guanidine hydrochloride solution, followed by 1 M dithiothreitol (DTT) to a final concentration of 20 mM. The mixture was incubated at 37°C for 90 min. After cooling to room temperature, 1 M iodoacetamide (final concentration 50 mM) was added, and the mixture was incubated at room temperature in the dark for 45 min. 2 M urea (Tris-HCl, pH 7.5) was added to the upper layer of a 10 kDa ultrafiltration membrane. The membrane was centrifuged at 13000 rpm for 10 min, and the waste liquid was discarded. This process was repeated twice. Trypsin was added at an enzyme:protein ratio of 1:25 (w:w), and the mixture was incubated at 37°C for 4 hours. After the reaction was completed, 0.5 μL of FA was added to the enzymatic hydrolysate to terminate the enzymatic hydrolysis. The supernatant was then collected by centrifugation for analysis.

[0132] The ultra-high performance liquid chromatograph (UHPLC) used was an ACQUITY UPLC H-Class PLUS (Waters), with the following parameters: column temperature 50℃, flow rate 0.3 mL / min, detection wavelength 214 nm, mobile phase A: 0.1% formic acid aqueous solution, and mobile phase B: 0.1% formic acid acetonitrile solution. The high resolution mass spectrometer (HPLC) used was a Vion Q-TOF (Waters), with MSE acquisition mode, capillary voltage (kV) 3V, MSE collision energy 20-45 eV, and ion source temperature 120℃. The raw HPLC data were analyzed using UNIFI software.

[0133] The percentage of modified sites is calculated using the following formula: Percent Q(X) = Area Q(X) / (Area(X) + Area Q(X)) × 100%, where Percent Q(X) is the percentage of amino acid sites X that have undergone modification. The detection results are shown in Table 2.

[0134] Table 2 Modification rate of ADC coupling sites

[0135] The results showed that the antibody-drug conjugates formed by enzymatically conjugating humanized antibodies hu17-H2L1, hu17-H3L1, and hu17-H4L1 with LND1002 achieved 100% modification rates at Q295 (EU numbering) of the antibody heavy chain HC and GGLQSGA at the carboxyl terminus of the antibody light chain LC.

[0136] Example 7: Determination of Antibody-Drug Conjugate Ratio (DAR) for ADCs

[0137] 1. UC961-vc MMAE(VLS101)

[0138] The distribution of drug antibody-drug conjugation ratio (DAR) was determined and the DAR value was calculated using hydrophobic chromatography-HPLC. Experimental Instruments: High-performance liquid chromatograph (e2695, Waters); Column: TSKgel Butyl-NPR, 4.6mm*10cm (catalog number 0042168 / TOSOH); Experimental Method:

[0139] Mobile phases: Mobile phase A (20 mM PB, pH 7.0, 1.5 M (NH4)2SO4 aqueous solution) and mobile phase B (20 mM PB, pH 7.0, 25% isopropanol). The flow rate was controlled at 0.8 mL / min, the column temperature at 30℃, and the detection wavelength at 280 nm. The gradient program was: 0-20 min, mobile phase B from 0-100%; 20-25 min, mobile phase B to 100%; 25-30 min, mobile phase A to 100%. Unlinked molecules were designated DAR0, molecules linked to 2 molecules were designated DAR2, molecules linked to 4 molecules were designated DAR4, molecules linked to 6 molecules were designated DAR6, and molecules linked to 8 molecules were designated DAR8. Therefore, elution was performed in the order of DAR0, DAR2, DAR4, DAR6, and DAR8. The experimental results were analyzed using the area normalization method, and the DAR value was approximately 3.9 (see Figure 3).

[0140] 2. Molecular weight verification of Hu17-H2L1-4LND1002, Hu17-H3L1-4LND1002, and Hu17-H4L1-4LND1002

[0141] Molecular weight was determined using liquid chromatography-mass spectrometry (LC-MS). The experimental method was as follows: A column (Column AdvanceBio SEC 200A, 1.9 μm, 2.1 × 15) was selected; mobile phase A was 100 mM ammonium formate, pH 7.0; isocratic elution was performed for 12 min. Mass spectrometry (6230 TOF, Agilent) parameters were set as follows: Mass Range 2000-8000 m / z, Vcap 3500 V, Dry Gas Temp 300℃, Dry Gas Flow 10 L / min, Sheath Gas Temp 325℃, Sheath Gas Flow 10 L / min. The experimental results are shown in Table 3.

[0142] Table 3: Detection of DAR values ​​for ND1002 of Hu17-H2L1, Hu17-H3L1, and Hu17-H4L1-4L.

[0143] The results showed that the antibody-drug conjugates formed by enzymatic conjugation of humanized antibodies hu17-H2L1, hu17-H3L1 and hu17-H4L1 with LND1002 had a DAR value of approximately 4.0.

[0144] Example 8: In vivo efficacy of ADC

[0145] 1. In vivo efficacy of H1975 human lung adenocarcinoma transplant tumor

[0146] In this experiment, age-appropriate female NUNU mice were inoculated with H1975 cells to construct a human lung adenocarcinoma H1975 xenograft model in nude mice. The tumor volume was increased to approximately 100 mm². 3 Twenty-eight animals with well-developed tumors were selected and divided into four groups based on tumor volume. A: Solvent control group (n=7), administered 0.9% sodium chloride injection (0.9% INJ NS, solvent control group); B: Positive control group (n=7), administered UC961-vc MMAE; C: Hu17-H2L1-4LND1002 experimental group (n=7); D: Hu17-H3L1-4LND1002 experimental group (n=7). All groups received the medication once a week for a total of two administrations: 3 mg / kg on day 0 and 4 mg / kg on day 7. Mice were weighed after administration, and data were recorded. Tumor growth was dynamically observed by measuring tumor diameter at different time points after administration. The experiment ended on day 20 (D20). Mice were asphyxiated with carbon dioxide, and the tumors were removed and weighed. The experimental results are shown in Table 4 and Figure 4. Tumor inhibition rate % = (Tumor weight in control group - Tumor weight in experimental group) / Tumor weight in control group * 100%

[0147] Table 4: In vivo efficacy of ADC against H1975 human lung adenocarcinoma xenografts Note: ***: P < 0.001; **: P < 0.01; *: P < 0.05

[0148] The results showed that the tumor inhibition rates (TWI) of the UC961-vc MMAE (VLS101), Hu17-H2L1-4LND1002, and Hu17-H3L1-4LND1002 groups were 51.0%, 65.8%, and 53.7%, respectively. Compared with the solvent control group, all groups significantly inhibited tumor growth. Compared with the positive control UC961-vc MMAE group, the Hu17-H2L1-4LND1002 group showed a more significant inhibitory effect on tumors, while the Hu17-H3L1-4LND1002 group had a comparable inhibitory effect on tumors to the positive control group.

[0149] 2. In vivo drug efficacy in HCC1187 human breast cancer transplant tumors

[0150] In this experiment, age-appropriate female NUNU mice were inoculated with HCC1187 cells to construct a human breast cancer HCC1187 xenograft model in nude mice. The tumor volume was increased to approximately 100 mm². 3 Twenty-eight animals with well-developed tumors were selected and divided into four groups based on tumor volume: A: Solvent control group (n=7), administered intravenously with 0.9% sodium chloride injection (0.9% INJ NS); B: Positive control group (n=7), administered with UC961-vc MMAE; C: Hu17-H2L1-4LND1002 experimental group (n=7); D: Hu17-H3L1-4LND1002 experimental group (n=7). All groups received 5 mg / kg of the drug at D0, D8, and D12 (administration on days 0, 8, and 12, for a total of three administrations). Mice were weighed after administration, and data were recorded. Tumor growth was dynamically observed by measuring tumor diameter at different time points after administration. The experiment ended on day 14. Mice were asphyxiated with carbon dioxide, and the tumors were removed and weighed. The results are shown in Table 5 and Figure 5.

[0151] Table 5: In vivo efficacy of ADC in HCC1187 human breast cancer xenografts Note: ***: P < 0.001; **: P < 0.01; *: P < 0.05

[0152] The results showed that the tumor inhibition rates of UC961-vc MMAE (VLS101), Hu17-H2L1-4LND1002, and Hu17-H3L1-4LND1002 were 38.1%, 55.7%, and 47.7%, respectively. Compared with the solvent control group, all three inhibited tumor growth. Compared with the positive control UC961-vc MMAE group, the experimental groups Hu17-H2L1-4LND1002 and Hu17-H3L1-4LND1002 showed more significant inhibitory effects.

[0153] Example 9: Screening of pH and buffer salts for liquid formulations

[0154] Based on the basic properties of this product, histidine-histidine hydrochloride, citrate-sodium citrate, and succinic acid-sodium hydroxide were selected as screening buffer salts with a buffer salt ion concentration of 20 mM. The pH was adjusted to 5.0, 5.5, 6.0, or 6.5 to prepare 12 solutions with different pH values ​​and buffer salt combinations. The Hu17-H2L1-4LND1002 drug conjugate sample after coupling reaction was ultrafiltered and replaced into the above 12 solutions to obtain Hu17-H2L1-4LND1002 solution. The final concentration of the Hu17-H2L1-4LND1002 drug conjugate was approximately 10 mg / mL. The solution was then subjected to high temperature (45℃±2℃) for 7 days (7d) and light exposure (5℃±3℃, white light: 5000 Lux±500 Lux, UV: 90 μW / cm²). 2 Stability test over 5 days (5d). Changes in the content of Hu17-H2L1-4LND1002 were determined using ultraviolet spectrophotometry (UV). Size exclusion chromatography (SEC-HPLC) was used to detect molecular size variants of Hu17-H2L1-4LND1002, and charge heterogeneity of Hu17-H2L1-4LND1002 was detected using cation exchange high performance liquid chromatography (CEX-HPLC).

[0155] Detection method:

[0156] UV: Zeroing was performed using a protein-free buffer solution. The absorbance of solutions with different pH values ​​and buffer salt combinations at 280 nm was measured using Nanodrop 2000. The protein content was calculated based on Beer-Lambert's law and the extinction coefficient (1.672).

[0157] SEC-HPLC: Following the high-performance liquid chromatography method in Section IV, General Chapter 0512 of the 2020 edition of the Chinese Pharmacopoeia, a chromatographic gel suitable for separating proteins with molecular weights of 10–500 kD was used as the stationary phase (e.g., [missing information]). 7.8×300mm, A chromatographic column (3.5 μm or other suitable column) was used. The mobile phase consisted of 100 mmol / L phosphate buffer, 100 mmol / L NaCl, and 10% isopropanol, with a pH of 6.7 ± 0.1. The flow rate was 0.8 mL / min, and the detection wavelength was 280 nm. An appropriate amount of the test solution was injected into the liquid chromatograph, and the ratio of protein monomer peaks and high molecular weight substances (HMWS) peaks to low molecular weight substances (LMWS) peaks was calculated using the area normalization method.

[0158] CEX-HPLC: Following the high-performance liquid chromatography method in General Chapter 0512 of the 2020 edition of the Chinese Pharmacopoeia, a cation exchange column was used. The mobile phase was: Phase A: 20 mM pH 6.1 sodium morpholine ethanesulfonate (containing 10% isopropanol; Phase B: 20 ​​mM pH 6.1 sodium morpholine ethanesulfonate 500 mM sodium chloride) (YMC, Biopro IEX SF 100×4.6 mm 5 μm). The detection wavelength was 280 nm. An appropriate amount of the test solution was injected into the liquid chromatograph, and the proportions of the acidic peak, main peak, and basic peak of the test sample were calculated using the area normalization method.

[0159] Test results:

[0160] Table 6: Results of Protein Content and Purity Detection Note: In the numbering, H represents histidine-histidine hydrochloride, C represents citric acid-sodium citrate, S represents succinic acid-sodium hydroxide, and the preceding number indicates the pH value.

[0161] Table 6 shows the experimental data on the effects of high temperature and light on the purity and protein content of Hu17-H2L1-4LND1002 under different types of buffer salts and pH values. After 7 days of high temperature, the protein concentration in the citrate-sodium citrate test groups at pH 5.0 and pH 5.5 decreased significantly, while no significant changes were observed in the protein concentration of other test groups. After 5 days of light exposure, no significant changes were observed in the protein concentration of all 12 test groups. In the same buffer solution, the SEC monomer in the pH 6.0 and pH 6.5 test groups exhibited the best high-temperature stability. At the same pH, the SEC monomer in the histidine-histidine hydrochloride test group exhibited the best high-temperature stability. In the same buffer solution, the CEX main peak in the pH 6.0 and pH 6.5 test groups exhibited the best high-temperature stability. At the same pH, the CEX main peak in the histidine-histidine hydrochloride test group showed the best light and high-temperature stability. Based on the combined data of protein concentration, SEC monomer, and CEX main peak, the histidine-histidine hydrochloride buffer system at pH 6.0–6.5 was determined to be the optimal buffer system. Since the optimal pH buffer range for histidine buffer is 5.5–6.5, a pH of 6.3 is preferred based on comprehensive evaluation.

[0162] Example 10: Screening of histidine concentration, stabilizer dosage, and protein concentration

[0163] Using an orthogonal design from the DOE design, with sucrose as the stabilizer for screening, according to L9(3 4 An orthogonal array was used to design a three-factor, three-level orthogonal experiment for histidine, sucrose, and protein concentrations, with a blank column used as a source of inter-group error. Table 7 shows the L9(3) orthogonal array designed for this experiment. 4An orthogonal array was used. Nine solution systems were prepared based on the orthogonal array. Hu17-H2L1-4LND1002 ultrafiltration was applied to these nine solution systems to dilute the protein concentration to the values ​​required by the orthogonal array. After filtration through a 0.22 μm microporous membrane, the samples were subjected to high temperature (45℃±2℃) for 14 days and light exposure (5℃±3℃, white light: 5000Lux±500Lux, UV: 90μW / cm²). 2 A 10-day stability test was conducted, detecting SEC monomers, the CEX main peak, and free MMAE. Table 8 shows the mean values ​​of the three experimental factors at different levels. Table 9 shows the p-values ​​and significance results for different experimental factors. For significantly influential factors, the optimal level can be selected; for non-significantly influential factors, all three levels can be chosen. The screening results for each detection indicator were comprehensively compared to select the optimal level for the three experimental factors. Table 10 summarizes the screening results for all items in the orthogonal experiment.

[0164] Table 7: L9(3) 4 Orthogonal array Note: The levels of each factor are set to 1, 2, and 3 from low to high. The fourth column of the orthogonal array is a blank column used to calculate the between-group error.

[0165] Detection methods

[0166] SEC-HPLC: The detection method is described in Example 9.

[0167] CEX-HPLC: The detection method is described in Example 9.

[0168] Free MMAE toxin: Based on the 2020 edition of the Chinese Pharmacopoeia, Part IV, General Chapter 0512 High Performance Liquid Chromatography and 0431 Mass Spectrometry, using octadecyl-bonded silica gel as the packing material (such as Waters ACQUITY). A BEH-C18 column (2.1 × 50 mm, particle size 1.7 μm or other suitable column) was used. Mobile phase A was an aqueous solution containing 0.1% TFA, and mobile phase B was an acetonitrile solution containing 0.1% TFA. The column temperature was 40℃, the flow rate was 0.4 mL / min, and the detection wavelength was 250 nm. 50 μL (10 mg / mL) of the test sample was taken, and internal standard working solution was added. Precipitation was performed with acetonitrile at a ratio of 1:3. The sample was placed at -20℃, in an ice bath for 60 min, centrifuged, and the supernatant was loaded. Different concentrations of MMAE were used as reference standards. After gradient elution by liquid chromatography, the sample was introduced into a mass spectrometer detector. Parameters such as the capillary and cone voltage, ion source temperature, collision energy, and nebulization flow rate were set before analysis. The free MMAE content in the test sample was calculated according to the standard curve. Free MMAE content (%) = MMAE detection concentration / ADC concentration × 100%

[0169] Table 8: Mean values ​​of different levels of each factor in orthogonal design

[0170] Table 9: P-values ​​and significance results for different experimental factors Note: " / " indicates not significant; "*" indicates significant; "**" indicates highly significant.

[0171] Table 10: Summary of Orthogonal Experiment Screening Results Note: "√" indicates an option; "×" indicates an option not to be selected.

[0172] Based on comprehensive analysis, the optimal levels for the three factors are: histidine concentration 30 mM, sucrose concentration 8% (w / v), and protein concentration 10 mg / mL.

[0173] Example 11: Stabilizer Screening

[0174] Based on the results of orthogonal design formulation screening in Example 10, a solution system containing 8% (w / v) sucrose or 8% (w / v) trehalose and a pH of 6.3, consisting of 30 mM histidine-histidine hydrochloride buffer, was prepared. Hu17-H2L1-4LND1002 ultrafiltration was used to replace the aforementioned buffer solution system containing both stabilizers. A buffer solution without stabilizers was used as a control. The protein concentration was adjusted to approximately 10 mg / mL. The protein was then subjected to high temperature (45℃±2℃) for 14 days and light exposure (5℃±3℃, white light: 5000 Lux±500 Lux, UV: 90 μW / cm²). 2 A 10-day stability test was conducted, and data such as SEC monomer, CEX main peak, and free MMAE content were measured. The results are shown in Table 11.

[0175] 20mL injection vials were used, and the vials were filled at a rate of 6.4mL / vial. Lyophilization was performed according to the laboratory platform's lyophilization process (equipment: Dongfulong LYO-7.5 lyophilizer; method: pre-freezing at -40℃ for 180min, single drying at -5℃ and 0.08mbar for 2200min, and desorption drying at 35℃ and 0.2mbar for 900min). The appearance, moisture content, SEC monomer content, CEX main peak, and free MMAE content of the lyophilized powder prepared with buffer solutions containing 8% (w / v) sucrose or 8% (w / v) trehalose stabilizer were compared. The results are shown in Table 12.

[0176] Detection method:

[0177] SEC-HPLC: The detection method is described in Example 9.

[0178] CEX-HPLC: The detection method is described in Example 9.

[0179] Free toxin MMAE: The detection method is described in Example 10.

[0180] Method for determining the moisture content of lyophilized powder: Karl Fischer method, using a Swiss Metrohm Karl Fischer moisture analyzer. Add anhydrous methanol solvent to the titration reaction vessel, immerse the electrode, and titrate with Karl Fischer reagent until the endpoint equilibrium is reached. Accurately weigh approximately 0.20-0.30 g of the test sample using the weight reduction method, add it to the titration reaction vessel, and perform titration. Calculate the moisture content of the lyophilized powder based on the volume of titrant consumed and the titration degree.

[0181] Table 11: Screening of Stabilizer Types (Comparison of Light and High Temperature Stability Data)

[0182] Table 12: Comparison of the quality of freeze-dried powders with 8% sucrose and 8% trehalose as stabilizers

[0183] Table 11 summarizes the data from the stabilizer screening. Samples containing 8% (w / v) sucrose and 8% (w / v) trehalose stabilizers showed similar stability data under high temperature and light conditions. At the same dosage, the two stabilizers did not significantly differ in the high temperature and light stability of the Hu17-H2L1-4LND1002 solution. After treatment at 45℃, the CEX peak of the Hu17-H2L1-4LND1002 solution decreased significantly, while the content of free toxin MMAE increased significantly, indicating that temperature had a significant impact on the stability of the Hu17-H2L1-4LND1002 solution. After light treatment, the CEX peak decreased slightly, and the content of free toxin MMAE increased slightly; compared to high temperature treatment, light treatment had a smaller impact on the stability of the Hu17-H2L1-4LND1002 solution. Figure 6 shows the morphological photographs of freeze-dried powders of Hu17-H2L1-4LND1002 (batch A sample) containing 8% (w / v) sucrose stabilizer and 8% (w / v) trehalose stabilizer. The freeze-dried powder containing 8% (w / v) sucrose stabilizer showed obvious shrinkage and collapse at the bottom of the cake, while the freeze-dried powder containing 8% (w / v) trehalose stabilizer did not show obvious shrinkage and collapse. Table 12 compares the mass of freeze-dried powders containing 8% (w / v) sucrose stabilizer and 8% (w / v) trehalose stabilizer. Both sugars were used as freeze-drying protectants for Hu17-H2L1-4LND1002. There was no significant difference in moisture content between the same batch of freeze-dried powders, and the purity of Hu17-H2L1-4LND1002 did not change significantly compared to the solution. Both stabilizers can be used as freeze-drying protectants for this product, with 8% (w / v) trehalose being the preferred freeze-drying protectant for Hu17-H2L1-4LND1002.

[0184] Example 12: Surfactant Dosage Screening

[0185] By analyzing the changes in the number of non-spherical particles (NSPs) in Hu17-H2L1-4LND1002 solution systems containing different contents of polysorbate 20 or polysorbate 80 after freeze-thaw and shaking tests, the optimal surfactant and its addition amount were screened. Hu17-H2L1-4LND1002 was ultrafiltered to a pH 6.3, 30 mM histidine-histidine hydrochloride, 8% (w / v) trehalose solution system, and the protein concentration was diluted to approximately 10 mg / mL. 0.005%, 0.02%, or 0.04% (w / v) of polysorbate 20 (PS20) or polysorbate 80 (PS80) were added, respectively, with the group without surfactant serving as a control. The solution was filtered through a 0.22 μm microporous membrane. Sub-visible particle data were measured using Flowcam after three freeze-thaw cycles (-20℃ to room temperature) and five days of shaking at 200 rpm at 5℃ ± 3℃. The results are shown in Table 13.

[0186] Table 13: Variation data of non-spherical subvisible particles (NSP) in freeze-thaw and shaking tests Note: Particle size (μm) in the table refers to the equivalent sphere diameter (ESD).

[0187] Table 13 shows the changes in non-spherical particles (NSPs) in solution systems with different amounts of surfactant after freeze-thaw and shaking tests. After three freeze-thaw cycles and five days of shaking, the number of NSPs in the unsurfactant group increased significantly, while no order-of-magnitude difference was observed in the six groups with surfactants. Based on comprehensive evaluation, the formulation of Hu17-H2L1-4LND1002 was selected to add 0.04% (w / v) polysorbate 20 as the surfactant.

[0188] Example 13: Development of Pre-freezing Process

[0189] Using the preferred formulation composition: 10 mg / mL Hu17-H2L1-4LND1002, 30 mM histidine-histidine hydrochloride, 0.04% (w / v) polysorbate 20, 8% (w / v) trehalose, pH 6.3 as the base formulation (i.e., the stock solution below), a freeze-drying process was developed (freeze dryer: Dongfulong, LYO 0.5). The glass transition temperature and collapse temperature of the sample with this formulation composition were detected by low-temperature DSC (manufacturer: Mettler Toledo, model: DSC3). The glass transition temperature of the sample was measured to be -30.61℃, and the collapse temperature of the sample was measured to be -26.4℃ by freeze-drying microscopy (manufacturer: BTL, model: Lyostat2).

[0190] Based on the glass transition temperature (-30.61℃) determined by the Hu17-H2L1-4LND1002 formulation solution, the proposed pre-freezing temperature for the small-scale test was -40℃. Before the pre-freezing cooling began, the plates were maintained at 4℃ for 30 min to ensure a uniform initial cooling temperature for the products. The freeze-drying effects of two cooling rates, 0.5℃ / min and 2℃ / min, were investigated. The freeze-drying formulation is shown in Table 14.

[0191] Table 14: Comparison of freeze-drying formulations for pre-freezing processes Note: Freeze dryer model: Dongfulong LYO-0.5, freeze drying capacity: 126 vials, filling volume: 65mg / bottle.

[0192] Table 15: Comparison of Moisture Content in Freeze-Dried Powders Processed in Pre-Freezing Process Note: a, b, and c represent three bottles of freeze-dried powder selected sequentially from the edge to the center of the plate, respectively.

[0193] The lyophilized powders obtained at different pre-freezing and cooling rates were all white, cake-like, loose powders. After injecting 6 mL of water for injection, both batches of lyophilized powder could be reconstituted into a clear liquid within 3 minutes. Table 15 shows the moisture content of three lyophilized powders containing Hu17-H2L1-4LND1002 at different locations. There was no significant difference in moisture content between the lyophilized powders obtained at the two pre-freezing rates. To save pre-freezing time, Hu17-H2L1-4LND1002 was pre-frozen at a cooling rate of 2℃ / min. During scale-up production, due to increased production batches, the cooling rate of the freeze dryer may not reach 2℃ / min. Therefore, during production, the cooling rate should be controlled at the fastest rate achievable by the equipment, typically between 1 and 2℃ / min.

[0194] Example 14: Development of a One-Step Drying Process

[0195] Pre-freezing was performed at a cooling rate of 2℃ / min, followed by analytical drying at 30℃ and a vacuum of 0.2 mbar for 1200 min. Two batches of freeze-dried samples with different primary drying parameters were compared. During the primary drying, the vacuum setting of the drying oven must be lower than the saturated vapor pressure of the product at a specific temperature for moisture to sublimate. Batch A and batch C were freeze-dried using two primary drying methods: high temperature and low pressure (-5℃, vacuum 0.08 mbar) and low temperature and high pressure (-20℃, vacuum 0.2 mbar), respectively, for 2200 min and 2400 min, respectively. The pressure rise values ​​for both batches were low (pressure rise < 0.01 mbar / min).

[0196] Table 16: Development parameters and moisture data for the primary drying process

[0197] Table 16 shows the pressure rise test and moisture data of the lyophilized powder under two primary drying parameters. Under the same lyophilization scale, the primary drying time was similar for both high-temperature low-pressure and low-temperature high-pressure primary drying parameters, and the resulting lyophilized powders were both white and loose (as shown in Figure 7). However, the low-temperature high-pressure lyophilized powder showed shrinkage and depressions at the bottom, while the high-temperature low-pressure lyophilized powder had a good morphology. In summary, the lyophilization of Hu17-H2L1-4LND1002 was carried out using a high-temperature low-pressure (-5℃, vacuum degree 0.08mbar) method for primary drying.

[0198] Example 15: Development of Desorption Drying Process

[0199] After the first drying cycle, approximately 10% moisture remains adsorbed on the capillary walls and polar groups of the dried material. When the bound water reaches a certain content, it provides conditions for the growth and reproduction of microorganisms and certain chemical reactions. This portion of moisture is bound water adsorbed onto the drug through weak molecular forces such as van der Waals forces and hydrogen bonds. To remove this moisture, a higher plate temperature is needed to transfer energy and overcome intermolecular forces, thus achieving the effect of removing the bound water. Pre-freezing was performed at a cooling rate of 2℃ / min, and the first drying cycle was performed at -5℃ with a vacuum of 0.08 mbar. The drying effect was analyzed by comparing the drying time at 30℃ with a vacuum of 0.2 mbar for 1200 min with that at 35℃ with a vacuum of 0.2 mbar for 900 min.

[0200] Table 17: Analysis of Drying Process Development Parameters and Moisture Data

[0201] Table 18: Comparison of mass of reconstituted solution and original solution of lyophilized powder obtained under different drying conditions

[0202] Table 17 compares the pressure rise test and moisture content of the lyophilized powder under two analytical drying conditions: 30℃ for 1200 min and 35℃ for 900 min. The moisture content of the lyophilized powder dried at 35℃ for 900 min is significantly lower than that dried at 30℃ for 1200 min. Table 18 compares the purity of the reconstituted solution and the original solution of the lyophilized powder obtained by the two analytical drying methods. Compared with the original solution, there is no significant difference in the purity of Hu17-H2L1-4LND1002 of the lyophilized powder obtained at the two analytical drying temperatures. In summary, the analytical drying process is determined to be 35℃, vacuum degree 0.20 mbar, and set time 900 min.

[0203] Example 16: Formulation stability

[0204] Two batches of samples (approximately 10 L of stock solution) were prepared under GMP conditions using an optimized formulation (10 mg / mL Hu17-H2L1-4LND1002, 30 mM histidine-histidine hydrochloride, 0.04% (w / v) polysorbate 20, 8% (w / v) trehalose, pH 6.3) and a lyophilization process (see Table 19). Stability studies were conducted on both batches. All methods for detecting the research indicators were validated and proven accurate and reliable. Stability studies were performed on the stock solution before lyophilization and the finished product after lyophilization at 5 ± 3 °C. The lyophilized powder was dissolved in 6.2 mL of water for injection before testing. The results are shown in Tables 20 and 21. The results indicate that the product quality is more stable after lyophilization than in the liquid state. Charge variants in the liquid state are more prone to generating acidic variants, and the lyophilized dosage form is more conducive to the long-term storage of this product.

[0205] Detection method:

[0206] SEC-HPLC: The detection method is described in Example 9.

[0207] icIEF: The assay was performed according to the determination of monoclonal antibody charge variants in General Chapter 3129 of the 2020 edition of the Chinese Pharmacopoeia, Part IV. Pre-focusing time: 1 min, pre-focusing voltage: 1500 V, focusing time: 8 min, focusing voltage: 3000 V. Based on the isoelectric point (PI) characteristics of different charge variants, they were separated by capillary electrophoresis (General Chapter 0542). The isoelectric point of the charge variants was determined and the relative percentage content was calculated.

[0208] DAR Distribution: According to the 2020 edition of the Chinese Pharmacopoeia, Part IV, General Chapter 0512, High Performance Liquid Chromatography (HPLC) was used. A Tskgel Butyl-NPR column was employed. Mobile phase A consisted of 25 mM PB (pH 7.0) and 1 M ammonium sulfate; mobile phase B consisted of 75% 25 mM PB (pH 7.0) and 25% isopropanol. The column temperature was 30℃, the flow rate was 0.5 ml / min, and the detection wavelength was 280 nm. An appropriate amount of the test solution was injected into the HPLC system for gradient elution, and the DAR value and DAR4 content were calculated.

[0209] Unconjugated antibody level: Same as the DAR distribution detection method, the DAR0 peak content is reported as the unconjugated antibody level.

[0210] Free MMAE content: The detection method is described in Example 10.

[0211] Table 19: Freeze-drying process used in GMP batches Note: This process is applicable to the Dongfulong LYO-7.5 freeze dryer, with a raw liquid volume of 10L SYS6005 freeze dryer and a batch size of 1500±300 pieces / batch.

[0212] Table 20 shows the stability results of key quality indicators for batch 1 of GMP.

[0213] Table 21 Stability Results of Key Quality Indicators for GMP Batch 2

[0214] The relevant amino acid sequences involved in this application are as follows:

[0215] The use of any and all embodiments or exemplary language (e.g., “such as”) provided herein is intended only to better illustrate the application and does not constitute a limitation on the scope of the application, unless otherwise required. The language in the specification should not be construed as indicating that any unclaimed element is necessary for carrying out the application.

[0216] All publications and patent applications referenced in this specification are incorporated herein by reference, as if each individual publication or patent application were specifically and individually indicated to be incorporated herein by reference. Furthermore, any theories, mechanisms, proofs, or findings described herein are intended to further enhance the understanding of this application and are not intended to limit this application in any way to such theories, mechanisms, proofs, or findings. Although this application has been shown and described in detail in the accompanying drawings and the foregoing description, this application should be considered illustrative rather than restrictive.

Claims

1. A pharmaceutical composition comprising an antibody-drug conjugate targeting ROR1, comprising (i) an antibody-drug conjugate targeting ROR1, (ii) a buffer, (iii) a stabilizer, and (iv) a surfactant.

2. The pharmaceutical composition according to claim 1, wherein, The antibody-drug conjugate targeting ROR1 has a structure represented by Formula I: A-(LD)d (Formula I), wherein, A represents an antibody or its antigen-binding fragment that targets ROR1; D represents the drug component, selected from microtubule inhibitors of MMAE or its analogues; L is the connecting sub-part, with one end connected to A and the other end connected to the drug part D; d is an integer or decimal from 1 to 12.

3. The pharmaceutical composition according to claim 1 or 2, wherein, The antibody targeting ROR1 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, both of which contain three CDRs. The heavy chain variable region includes: CDR1-H, which has the amino acid sequence shown in SEQ ID NO:1; CDR2-H, having an amino acid sequence selected from those shown in SEQ ID NO:2, 9, and 11; and CDR3-H, which has the amino acid sequence shown in SEQ ID NO:3; The light chain variable region includes: CDR1-L has the amino acid sequence shown in SEQ ID NO:4; CDR2-L, which has the amino acid sequence shown in SEQ ID NO:5; and CDR3-L has the amino acid sequence shown in SEQ ID NO:

6.

4. The pharmaceutical composition according to any one of claims 1-3, wherein, The buffer is selected from histidine buffers, citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, and phosphate buffers; preferably, the buffer is a histidine buffer; more preferably, the histidine buffer is histidine-histidine hydrochloride.

5. The pharmaceutical composition according to any one of claims 1-4, wherein, The stabilizer is selected from sucrose, trehalose, mannitol, sorbitol, L-serine, monosodium glutamate, alanine, glycine, sarcosine, proline, and methionine; preferably trehalose.

6. The pharmaceutical composition according to any one of claims 1-5, wherein, The surfactant is polysorbate or poloxamer; preferably, the surfactant is selected from polysorbate 80, polysorbate 20 and poloxamer 188.

7. The pharmaceutical composition according to any one of claims 1-6, wherein, (a) The buffer is selected from histidine buffers, citrate buffers and succinate buffers; preferably, the buffer is a histidine buffer; more preferably, the histidine buffer is histidine-histidine hydrochloride; (b) The stabilizer is selected from sucrose and trehalose; preferably, the stabilizer is trehalose; (c) The surfactant is selected from polysorbate 80 and polysorbate 20; preferably, the surfactant is polysorbate 20.

8. The pharmaceutical composition according to any one of claims 1-7, wherein, The pharmaceutical composition is prepared in a form selected from solid dosage forms, semi-solid dosage forms, liquid dosage forms, and gaseous dosage forms; preferably, the pharmaceutical composition is prepared in the form of a liquid dosage form or a solid dosage form; preferably, the liquid dosage form is an aqueous injection; more preferably, the pharmaceutical composition is prepared in the form of a solid dosage form, preferably a lyophilized powder for injection.

9. The pharmaceutical composition according to any one of claims 1-8, wherein, The pharmaceutical composition is prepared as a liquid formulation, preferably an aqueous injectable formulation, wherein the liquid formulation contains: 5.0–15.0 mg / mL of an antibody-drug conjugate targeting ROR1, 20.0–40.0 mM of histidine-histidine hydrochloride, 5.0–10.0% (w / v) trehalose, 0.02–0.06% (w / v) polysorbate 20, and pH 6.0–6.

6.

10. The pharmaceutical composition according to any one of claims 1-9, wherein, The pharmaceutical composition is prepared as a liquid formulation, preferably an aqueous injectable formulation, wherein the liquid formulation contains: 9.0–11.0 mg / mL of an antibody-drug conjugate targeting ROR1, 28.0–32.0 mM of histidine-histidine hydrochloride, 7.0–9.0% (w / v) trehalose, 0.03–0.05% (w / v) polysorbate 20, and pH 6.0–6.

6.

11. The pharmaceutical composition according to any one of claims 1-10, wherein, The pharmaceutical composition is prepared as a liquid formulation, preferably an aqueous injectable formulation, the liquid formulation containing: about 10.0 mg / mL of an antibody-drug conjugate targeting ROR1, about 30.0 mM of histidine-histidine hydrochloride, about 8.0% (w / v) of trehalose, about 0.04% (w / v) of polysorbate 20, and a pH of about 6.

3.

12. The pharmaceutical composition according to any one of claims 1-11, wherein, The liquid formulation further contains water; preferably, the water is water for injection.

13. The pharmaceutical composition according to any one of claims 1-8, wherein, The pharmaceutical composition is prepared as a solid dosage form, preferably a lyophilized powder for injection, wherein the solid dosage form contains: 5.0% to 15.0% (wt%) of an antibody-drug conjugate targeting ROR1, 2.0% to 4.0% (wt%) of histidine, 1.0% to 3.0% (wt%) of histidine hydrochloride, 70.0% to 90.0% (wt%) of trehalose, and 0.01% to 1.00% (wt%) of polysorbate 20.

14. The pharmaceutical composition according to any one of claims 1-8, wherein, The pharmaceutical composition is prepared as a solid dosage form, preferably a lyophilized powder for injection, wherein the solid dosage form contains: 9.0% to 12.0% (wt%) of an antibody-drug conjugate targeting ROR1, 2.8% to 3.4% (wt%) of histidine, 2.0% to 2.5% (wt%) of histidine hydrochloride, 80.0% to 80.5% (wt%) of trehalose, and 0.30% to 0.50% (wt%) of polysorbate 20.

15. The pharmaceutical composition according to any one of claims 1-8, wherein, The pharmaceutical composition is prepared as a solid dosage form, preferably a lyophilized powder for injection, the solid dosage form containing: about 10.46% (wt%) of an antibody-drug conjugate targeting ROR1, about 3.24% (wt%) of histidine, about 2.20% (wt%) of histidine hydrochloride, about 83.68% (wt%) of trehalose, and about 0.42% (wt%) of polysorbate 20.

16. The pharmaceutical composition according to any one of claims 1-15, wherein, The pharmaceutical composition is a liquid preparation with pH = 6.0 to 6.6, preferably an aqueous injection; or a solid preparation obtained by lyophilizing a liquid preparation with pH = 6.0 to 6.6, preferably an aqueous injection, preferably a lyophilized powder for injection.

17. The pharmaceutical composition according to any one of claims 13-16, wherein, The lyophilized powder injection is prepared by a pre-freezing step, a single drying step, and a desorption drying step.

18. The pharmaceutical composition according to any one of claims 2-17, wherein, The antigen-binding fragment targeting ROR1 is a Fab fragment, an F(ab')2 fragment, or a single-chain Fv fragment (scFv).

19. The pharmaceutical composition according to any one of claims 3-18, wherein, The amino acid sequence of the heavy chain variable region is selected from the amino acid sequences shown in SEQ ID NO.7, SEQ ID NO.10 and SEQ ID NO.12, and the amino acid sequence of the light chain variable region is the amino acid sequence shown in SEQ ID NO.

8.

20. The pharmaceutical composition according to any one of claims 2-19, wherein, The heavy chain of the antibody comprises a heavy chain constant region of the amino acid sequence shown in SEQ ID NO. 13, and the light chain of the antibody comprises a light chain constant region of the amino acid sequence shown in SEQ ID NO.

19.

21. The pharmaceutical composition according to any one of claims 2-20, wherein, The C-terminus of the light chain of the antibody or its antigen-binding fragment is attached to a glutamine-containing tag peptide, preferably selected from LQSGA, GGLQSGA and GGGLQSGA.

22. The pharmaceutical composition according to any one of claims 2-21, wherein, The microtubule inhibitors of the drug fraction MMAE or its analogues are selected from auristatin E (AE), auristatin F (AF), monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), monomethylauristatin D (MMAD), dolastatin, and auristatin E5-benzoylvalerate (AEVB).

23. The pharmaceutical composition according to any one of claims 2-22, wherein, The L-connector portion is independently selected from -mc-Val-Cit-pAB-, -mc-Val-Cit-pABC-, -mc-Val-Cit-, and -NH-(CH2-CH2-O). m -Val-Cit- and -NH-(CH2-CH2-O) m -Val-Cit-pABC-, and -NH-(CH2-CH2-O) m -CH2-C(=O)-Val-Cit-pABC-, where -(CH2-CH2-O) m In the -, m is an integer from 1 to 8; preferably, m is 3.

24. The pharmaceutical composition according to any one of claims 1-23, wherein, The antibody-drug conjugate targeting ROR1 has a structure represented by Formula II: Preferably, A is selected from antibodies Hu17-H2L1, Hu17-H3L1, and Hu17-H4L1, and d is selected from integers or decimals from 1 to 8.

25. The pharmaceutical composition according to any one of claims 1-24, wherein, The antibody-drug conjugate targeting ROR1 is selected from: Hu17-H2L1-4LND1002: Hu17-H3L1-4LND1002: Hu17-H4L1-4LND1002:

26. The pharmaceutical composition according to any one of claims 1-25, wherein, The antibody contains Q295 in its heavy chain constant region, and the linker portion is linked to the side chain of the Q295 via an amide bond, and / or the linker portion is linked to the side chain of the glutamine residue in the glutamine-containing tag peptide via an amide bond.

27. The pharmaceutical composition according to any one of claims 8-12, wherein, The pharmaceutical composition is administered via intravenous infusion.

28. A method of treating or preventing cancer in an individual, comprising administering a therapeutically effective amount of the pharmaceutical composition of any one of claims 1 to 27 to the individual suffering from said cancer.

29. Use of the pharmaceutical composition according to any one of claims 1 to 27 in the preparation of a medicament for treating or preventing cancer.

30. The method according to claim 28 or the application according to claim 29, wherein, The cancer is a solid tumor, which is gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, or gallbladder cancer, preferably adenocarcinoma of the stomach, esophagus, pancreatic duct, bile duct, lung, or ovary; more preferably gastric cancer or pancreatic cancer.

31. The method according to claim 28 or the application according to claim 29, wherein, The cancer is a hematologic malignancy, preferably lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, non-Hodgkin's lymphoma, Richter-transformed non-Hodgkin's lymphoma, T-cell leukemia, Burkitt's lymphoma, multiple myeloma, marginal zone lymphoma, small lymphocytic lymphoma, marginal zone lymphoma, or marginal cell B-cell lymphoma.

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