Method for ameliorating or treating malignant ascites using Anti-epcam × CD3 bispecific antibody

Abstract

The present disclosure relates to a method for ameliorating or treating malignant ascites using an anti-EpCAM × CD3 bispecific antibody and a composition thereof and use of the anti-EpCAM × CD3 bispecific antibody and a composition thereof in ameliorating or treating malignant ascites. The present disclosure features better dosing convenience, longer dosing intervals, longer dosing cycles, and improved efficacy and safety.

Description

Method for improving or treating malignant ascites using anti-EpCAM×CD3 bispecific antibody Technical Field

[0001] The present invention relates to the field of immunology, and particularly to a method for improving or treating malignant ascites using a bispecific antibody. Background Art

[0002] EpCAM is a highly expressed antigen on the surface of epithelial tumor cells, which are the majority of tumors. In epithelial tumors such as ovarian cancer and gastrointestinal tract cancers, EpCAM is expressed in almost 100% of cases. CD3 is a T cell differentiation antigen and the first signal for activation.

[0003] Bispecific antibodies targeting EpCAM and CD3 primarily bridge immune cells and tumor cells through dual binding of EpCAM and CD3. After bridging, T cells are activated by CD3 signals and then target EpCAM to exert immune killing effects on tumor cells. Furthermore, anti-EpCAM × CD3 bispecific antibodies can block EpCAM downstream signaling and inhibit tumor growth, promote T cell activation and proliferation by binding to CD3, and enhance killing of EpCAM-positive cells through antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

[0004] Malignant ascites (MA) is an accumulation of fluid in the abdominal cavity caused by various malignant solid tumors that may involve the abdominal cavity, and occurs in up to 50% of patients with advanced cancer. MA is one of the indicators of poor prognosis for cancer patients. Depending on the primary malignant tumor, the average survival time after diagnosis is as low as 1 to 4 months. Due to the adverse effects of malignant effusion on the gastrointestinal and urogenital tracts, MA patients are often hospitalized due to symptoms such as abdominal pain, loss of appetite, difficulty breathing, constipation, frequent urination, anorexia and vomiting, and their quality of life is extremely poor. To date, there is no radical treatment for MA, and the main goal of treatment is to relieve MA symptoms. Currently, only Japan has issued guidelines for MA, which recommend drainage and diuretics as treatment methods; China and the United States do not yet have guidelines for MA.

[0005] Currently, there is no drug that can cure malignant ascites. To date, the only drug approved to relieve MA symptoms is Catumaxomab (anti-EpCAM×CD3 bispecific antibody), which was first approved for marketing by the European Medicines Agency (EMA) in 2009. However, there is still a clinical need for malignant ascites treatment options that are more convenient to administer, can be used for a long time, have better ascites control efficacy, better prolong survival, and are safe to administer.

[0006] The primary diseases of MA are most commonly ovarian cancer and gastrointestinal tumors (such as gastric cancer and colorectal cancer). In addition, MA can be caused by pancreatic cancer, liver cancer, uterine cancer, etc., and can also be caused by extra-abdominal malignancies such as breast cancer, lung cancer and lymphoma. Another 15 to 20% of patients have unknown primary lesions. Therefore, MA is a complication of malignant tumors with a large number of cases, serious life-threatening conditions, affecting the quality of life of patients, poor efficacy of existing treatments, and a serious lack of drugs and normative clinical research basic data. There is an urgent need for treatment options with better administration convenience, longer dosing intervals, longer dosing cycles, and better efficacy and safety. Summary of the Invention

[0007] Overall, the present invention relates to a method or use of an anti-EpCAMxCD3 diabody for alleviating or treating malignant ascites caused by malignant tumors. The dosing regimen includes an initial treatment period and a maintenance treatment period, optionally including pretreatment before administration, and has better administration convenience, longer dosing intervals, longer dosing cycles, and better efficacy and safety.

[0008] The present inventors have discovered that anti-EpCAM×CD3 bispecific antibodies, i.e., anti-EpCAM and CD3 bispecific antibodies, can, for example, be administered intraperitoneally to activate immune cells to clear and inhibit the growth and survival of tumor cells in the peritoneal cavity, reduce the blockage of lymphatic vessels by tumor cells, and reduce the secretion of inflammatory factors by tumor cells, thereby improving the symptoms of malignant ascites.

[0009] In one aspect, a method for improving and treating malignant ascites in a subject is provided, the method comprising:

[0010] 1) in the initial treatment period, administering the anti-EpCAM×CD3 bispecific antibody four times, wherein the first administration dose is about 10 to 50 μg, preferably about 50 μg, and the second, third, and fourth administration doses are about 50 to 400 μg, preferably about 400 μg each, and the subject undergoes ascites drainage before each administration, and;

[0011] 2) during the maintenance treatment period, administering the anti-EpCAM×CD3 bispecific antibody once every 2 weeks, with each dose ranging from about 50 to 400 μg, and preferably about 400 μg;

[0012] The anti-EpCAM×CD3 bispecific antibody is in a form suitable for intraperitoneal perfusion.

[0013] In another aspect, an anti-EpCAM×CD3 bispecific antibody is provided for improving or treating malignant ascites in a subject, wherein the method for improving or treating malignant ascites comprises:

[0014] 1) in the initial treatment period, administering the anti-EpCAM×CD3 bispecific antibody four times, wherein the first administration dose is about 10 to 50 μg, preferably about 50 μg, and the second, third, and fourth administration doses are about 50 to 400 μg, preferably about 400 μg each, and the subject undergoes ascites drainage before each administration, and;

[0015] 2) during the maintenance treatment period, administering the anti-EpCAM×CD3 bispecific antibody once every 2 weeks, with each dose ranging from about 50 to 400 μg, and preferably about 400 μg;

[0016] The anti-EpCAM×CD3 bispecific antibody is in a form suitable for intraperitoneal perfusion.

[0017] In some embodiments, the anti-EpCAM×CD3 bispecific antibody comprises (a) a light chain-heavy chain pair that specifically binds to EpCAM, wherein the light chain is bound to the heavy chain through a disulfide bond; and (b) a fusion peptide that specifically binds to CD3, wherein the fusion peptide comprises a single-chain variable fragment (scFv) and an Fc fragment having a CH2 domain and a CH3 domain, and the fusion peptide is bound to the heavy chain through a disulfide bond.

[0018] In some embodiments, the subject is pretreated by administering an antipyretic analgesic before the first and second administrations of the bispecific antibody during the initial treatment period. Thereafter, if the subject does not experience an infusion reaction and / or cytokine release syndrome during or after the administration of the bispecific antibody, there is no need to administer an antipyretic analgesic before the next administration of the bispecific antibody. If the subject experiences an infusion reaction and / or cytokine release syndrome during or after the administration of the bispecific antibody, an antipyretic analgesic is administered before the next administration of the bispecific antibody.

[0019] In some embodiments, the analgesic and antipyretic drug is an antihistamine, a steroidal and / or nonsteroidal drug, such as a glucocorticoid, for example, dexamethasone, diphenhydramine, or acetaminophen.

[0020] In some embodiments, the pretreatment is intraperitoneal infusion of about 5 mg of dexamethasone or about 20 mg of diphenhydramine.

[0021] In some embodiments, during the initial treatment period, the four administrations are respectively on day 1, day 4 or day 4±2 (i.e., 1-5 days apart from the first administration), day 11 or day 11±3 (i.e., 4-10 days apart from the second administration), and day 18 or day 18±4 (i.e., 3-11 days apart from the third administration), and the dosages administered are preferably about 50 μg, 400 μg, 400 μg, and 400 μg, respectively.

[0022] In some embodiments, after drainage is completed and before the antibody is administered, the subject is intraperitoneally perfused with saline, preferably about 100 to 650 ml of saline, and after the antibody is administered, the subject is intraperitoneally perfused with saline, preferably about 0 to 250 ml of saline.

[0023] In some embodiments, the anti-EpCAM×CD3 bispecific antibody is a solution comprising 0.01 to 5 mg / mL, preferably 0.5 mg / mL, of the anti-EpCAM×CD3 bispecific antibody, 30 mM histidine and histidine hydrochloride buffer, 5.0% (w / v) trehalose and 0.05% (w / v) PS80, and having a pH of 5.2.

[0024] In some embodiments, the administration volume of the anti-EpCAM×CD3 bispecific antibody is 100 to 250 ml (preferably, the anti-EpCAM×CD3 bispecific antibody is diluted with physiological saline to a volume of 100 to 250 ml), and preferably, the administration time of the anti-EpCAM×CD3 bispecific antibody is 0.5 to 3 hours.

[0025] In some embodiments, in the EpCAM×CD3 bispecific antibody,

[0026] The heavy chain-light chain pair that specifically binds to EpCAM comprises:

[0027] (i) CDRH1, CDRH2, and CDRH3 contained in the heavy chain variable region shown in SEQ ID NO: 14, and

[0028] (ii) CDRL1, CDRL2, and CDRL3 contained in the light chain variable region shown in SEQ ID NO: 13,

[0029] Preferably, the sequence of CDRL1 is shown in SEQ ID NO: 25, the sequence of CDRL2 is shown in SEQ ID NO: 26, the sequence of CDRL3 is shown in SEQ ID NO: 27, the sequence of CDRH1 is shown in SEQ ID NO: 28, the sequence of CDRH2 is shown in SEQ ID NO: 29, and the sequence of CDRH3 is shown in SEQ ID NO: 30; or

[0030] (iii) CDRH1, CDRH2, and CDRH3 contained in the heavy chain variable region set forth in SEQ ID NO: 16, and (iv) CDRL1, CDRL2, and CDRL3 contained in the light chain variable region set forth in SEQ ID NO: 15,

[0031] Preferably, the sequence of CDRL1 is shown as SEQ ID NO:31, the sequence of CDRL2 is shown as SEQ ID NO:32, the sequence of CDRL3 is shown as SEQ ID NO:33, the sequence of CDRH1 is shown as SEQ ID NO:34, the sequence of CDRH2 is shown as SEQ ID NO:35, and the sequence of CDRH3 is shown as SEQ ID NO:36;

[0032] The fusion peptide that specifically binds to CD3 comprises:

[0033] (i) CDRH1, CDRH2, and CDRH3 contained in the heavy chain variable region set forth in SEQ ID NO:43, and CDRL1, CDRL2, and CDRL3 contained in the light chain variable region set forth in SEQ ID NO:44,

[0034] Preferably, the sequence of CDRH1 is shown in SEQ ID NO:37, the sequence of CDRH2 is shown in SEQ ID NO:38, and the sequence of CDRH3 is shown in SEQ ID NO:39, the sequence of CDRL1 is shown in SEQ ID NO:40, the sequence of CDRL2 is shown in SEQ ID NO:41, and the sequence of CDRL3 is shown in SEQ ID NO:42; or

[0035] (ii) CDRH1, CDRH2, and CDRH3 contained in the heavy chain variable region set forth in SEQ ID NO:51, and CDRL1, CDRL2, and CDRL3 contained in the light chain variable region set forth in SEQ ID NO:52,

[0036] Preferably, the sequence of CDRH1 is shown as SEQ ID NO:45, the sequence of CDRH2 is shown as SEQ ID NO:46, and the sequence of CDRH3 is shown as SEQ ID NO:47, the sequence of CDRL1 is shown as SEQ ID NO:48, the sequence of CDRL2 is shown as SEQ ID NO:49, and the sequence of CDRL3 is shown as SEQ ID NO:50.

[0037] In some embodiments, in the anti-EpCAM×CD3 bispecific antibody,

[0038] The heavy chain-light chain pair that specifically binds to EpCAM comprises:

[0039] (i) a heavy chain variable region having a sequence as set forth in SEQ ID NO: 14, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical thereto, and a light chain variable region having a sequence as set forth in SEQ ID NO: 13, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical thereto; or

[0040] (ii) a heavy chain variable region having a sequence as set forth in SEQ ID NO: 16, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical thereto, and a light chain variable region having a sequence as set forth in SEQ ID NO: 15, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical thereto; and

[0041] The fusion peptide that specifically binds to CD3 comprises:

[0042] (i) a heavy chain variable region having a sequence as set forth in SEQ ID NO:43, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and a light chain variable region having a sequence as set forth in SEQ ID NO:44, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, or

[0043] (ii) a heavy chain variable region having a sequence as set forth in SEQ ID NO:51, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and a light chain variable region having a sequence as set forth in SEQ ID NO:52, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0044] Preferably, the anti-EpCAM×CD3 bispecific antibody comprises a heavy chain variable region having the sequence of SEQ ID NO: 14, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; a light chain variable region having the sequence of SEQ ID NO: 13, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; and a fusion peptide that specifically binds to CD3 comprising the sequence of SEQ ID NO: 18 or SEQ ID NO: 19, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0045] The present invention also relates to the following items:

[0046] 1. Use of an anti-EpCAM×CD3 bispecific antibody in the preparation of a medicament or kit for improving malignant ascites in a patient, wherein the anti-EpCAM×CD3 bispecific antibody is in a form suitable for intraperitoneal perfusion (preferably in the form of a lyophilized preparation), and the medicament or kit further comprises a product instruction sheet, wherein the product instruction sheet indicates a dosing regimen for the anti-EpCAM×CD3 bispecific antibody, and the dosing regimen comprises an initial treatment period and a maintenance treatment period, wherein the initial treatment period comprises four administrations of the anti-EpCAM×CD3 bispecific antibody, with the first administration dose being 10 to 50 μg (preferably 50 μg), the second, third, and fourth administration doses being 50 to 400 μg / time (preferably 400 μg / time), and the maintenance treatment period dose being 50 to 400 μg / time (preferably 400 μg / time). For the anti-EpCAM×CD3 bispecific antibody, preferably, the patient is pretreated before the first and second administrations. When the patient does not experience an allergic reaction and / or cytokine release syndrome during or after subsequent administration, the patient does not need to be pretreated before subsequent administration. If the patient experiences an allergic reaction and / or cytokine release syndrome reaction during or after subsequent administration, the patient is pretreated before subsequent administration. The pretreatment is to administer antipyretic, analgesic and / or antiallergic drugs (such as acetaminophen, dexamethasone sodium phosphate injection or diphenhydramine) to the patient. Preferably, the antipyretic, analgesic and / or antiallergic drugs are administered intravenously, orally or intraperitoneally 30 minutes before administration (more preferably, the intraperitoneal dosage of dexamethasone is 5 mg and the intraperitoneal dosage of diphenhydramine is 20 mg).

[0047] 2. The use described in item 1, wherein the initial treatment period comprises administering 50 μg, 400 μg, 400 μg, 400 μg on day 1, day 4 or day 4±2, day 11 or day 11±3, and day 18 or day 18±4, respectively, preferably draining the patient's ascites (preferably adequate drainage) before the first and second doses, or draining the patient's ascites (preferably adequate drainage) before each dose during the initial treatment period.

[0048] 3. The use according to any one of items 1-2, wherein during the initial treatment period, before administering the anti-EpCAM×CD3 bispecific antibody, the patient's peritoneal cavity is perfused with normal saline (preferably 100-650 ml), and after administering the anti-EpCAM×CD3 bispecific antibody, the patient's peritoneal cavity is perfused with normal saline (preferably 0-250 ml).

[0049] 4. The use according to any one of items 1 to 3, wherein in the initial treatment period, before administering the anti-EpCAM×CD3 bispecific antibody, 100-650 ml of normal saline is perfused into the patient's peritoneal cavity, and after administering the anti-EpCAM×CD3 bispecific antibody, normal saline (preferably 0-250 ml) is perfused into the patient's peritoneal cavity, wherein the anti-EpCAM×CD3 bispecific antibody is a solution of 50-400 μg of a lyophilized preparation dissolved in 100-250 ml (preferably 250 ml) of normal saline, and the administration time of the anti-EpCAM×CD3 bispecific antibody is preferably 0.5-3 hours.

[0050] 5. The use according to any one of items 1 to 4, wherein the maintenance treatment period comprises administering the anti-EpCAM×CD3 bispecific antibody once every 1 or 2 weeks after 18 days of administration, preferably wherein the anti-EpCAM×CD3 bispecific antibody is a solution of 400 μg of a lyophilized preparation dissolved in 250 ml of normal saline, and preferably the administration time of the anti-EpCAM×CD3 bispecific antibody is 0.5-3 hours.

[0051] 6. The use according to any one of items 1 to 5, wherein 5 mg of dexamethasone sodium phosphate injection is administered intramuscularly, intravenously, or intraperitoneally 30 minutes before the first and second doses of the drug during the initial treatment period; or 20 mg of diphenhydramine injection is administered intramuscularly or intraperitoneally; if no infusion reaction occurs during the period, pretreatment can be stopped for the third and subsequent doses.

[0052] The initial treatment period of the anti-EpCAM×CD3 bispecific antibody: 50 μg is administered by intraperitoneal perfusion for the first treatment, followed by intraperitoneal perfusion on day 4 or day 4±2, day 11 or day 11±3, and day 18 or day 18±4, 400 μg each time. Before each administration, the ascites is fully drained, and 500 ml of normal saline and the anti-EpCAM×CD3 bispecific antibody diluted with 250 ml of normal saline are sequentially perfused. After the end of the administration, 0-250 ml of normal saline is administered by intraperitoneal perfusion.

[0053] Maintenance treatment period of the anti-EpCAM×CD3 bispecific antibody: After treatment on day 18 or day 18±4, 400 μg is administered once every 7 days or every 14 days. During the maintenance treatment period, ascites drainage is no longer performed before administration, and normal saline intraperitoneal perfusion is no longer performed before and after administration.

[0054] Preferably, the intraperitoneal infusion time of the anti-EpCAM×CD3 bispecific antibody is 0.5-3 hours.

[0055] 7. The use according to any one of items 1 to 6, wherein the EpCAM×CD3 bispecific antibody comprises an antigen-binding domain that specifically binds to EpCAM and an antigen-binding domain that specifically binds to CD3,

[0056] The antigen-binding domain that specifically binds to EpCAM is selected from the group consisting of:

[0057] 1) An antigen-binding domain that specifically binds to EpCAM and comprises the following CDRs or variants thereof:

[0058] (i) CDRH1, CDRH2 and CDRH3 contained in the heavy chain variable region shown in SEQ ID NO: 14, and

[0059] (ii) CDRL1, CDRL2 and CDRL3 contained in the light chain variable region shown in SEQ ID NO: 13,

[0060] Preferably, the sequence of CDRL1 is shown in SEQ ID NO: 25, the sequence of CDRL2 is shown in SEQ ID NO: 26, the sequence of CDRL3 is shown in SEQ ID NO: 27, the sequence of CDRH1 is shown in SEQ ID NO: 28, the sequence of CDRH2 is shown in SEQ ID NO: 29, and the sequence of CDRH3 is shown in SEQ ID NO: 30; or

[0061] 2) an antigen-binding domain that specifically binds to EpCAM and comprises the following CDRs or variants thereof:

[0062] (i) CDRH1, CDRH2 and CDRH3 contained in the heavy chain variable region shown in SEQ ID NO: 16, and

[0063] (ii) CDRL1, CDRL2 and CDRL3 contained in the light chain variable region shown in SEQ ID NO: 15,

[0064] Preferably, the sequence of CDRL1 is shown as SEQ ID NO:31, the sequence of CDRL2 is shown as SEQ ID NO:32, the sequence of CDRL3 is shown as SEQ ID NO:33, the sequence of CDRH1 is shown as SEQ ID NO:34, the sequence of CDRH2 is shown as SEQ ID NO:35, and the sequence of CDRH3 is shown as SEQ ID NO:36;

[0065] The antigen binding domain that specifically binds to CD3 is selected from the group consisting of:

[0066] 1) An antigen-binding domain that specifically binds to CD3 comprising the following CDRs or variants thereof:

[0067] CDRH1, CDRH2 and CDRH3 contained in the heavy chain variable region shown in SEQ ID NO:43, and CDRL1, CDRL2 and CDRL3 contained in the light chain variable region shown in SEQ ID NO:44,

[0068] Preferably, the sequence of CDRH1 is shown in SEQ ID NO:37, the sequence of CDRH2 is shown in SEQ ID NO:38, and the sequence of CDRH3 is shown in SEQ ID NO:39, the sequence of CDRL1 is shown in SEQ ID NO:40, the sequence of CDRL2 is shown in SEQ ID NO:41, and the sequence of CDRL3 is shown in SEQ ID NO:42; or

[0069] 2) an antigen-binding domain that specifically binds to CD3 comprising the following CDRs or variants thereof:

[0070] CDRH1, CDRH2 and CDRH3 contained in the heavy chain variable region shown in SEQ ID NO:51, and CDRL1, CDRL2 and CDRL3 contained in the light chain variable region shown in SEQ ID NO:52,

[0071] Preferably, the sequence of CDRH1 is as shown in SEQ ID NO:45, the sequence of CDRH2 is as shown in SEQ ID NO:46, and the sequence of CDRH3 is as shown in SEQ ID NO:47, the sequence of CDRL1 is as shown in SEQ ID NO:48, the sequence of CDRL2 is as shown in SEQ ID NO:49, and the sequence of CDRL3 is as shown in SEQ ID NO:50; wherein the variant CDRs have 3, 2 or 1 amino acid differences, respectively, or have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, respectively, to the corresponding CDRs,

[0072] Preferably, the antigen-binding domain that specifically binds to EpCAM is in the form of a Fab fragment, and the antigen-binding domain that specifically binds to CD3 is in the form of an ScFv.

[0073] 8. The use according to any one of items 1 to 7, wherein the antigen-binding domain that specifically binds to EpCAM comprises the following heavy chain variable region and light chain variable region (or variants thereof):

[0074] (i) the heavy chain variable region set forth in SEQ ID NO: 14, and the light chain variable region set forth in SEQ ID NO: 13; or

[0075] (ii) the heavy chain variable region set forth in SEQ ID NO: 16, and the light chain variable region set forth in SEQ ID NO: 15; and

[0076] The antigen-binding domain that specifically binds to CD3 comprises the following heavy chain variable region and light chain variable region (or variants thereof):

[0077] (1) the heavy chain variable region shown in SEQ ID NO: 43 and the light chain variable region shown in SEQ ID NO: 44, or

[0078] (2) the heavy chain variable region set forth in SEQ ID NO:51 and the light chain variable region set forth in SEQ ID NO:52;

[0079] Preferably, the antigen binding domain that specifically binds to EpCAM comprises the following heavy chain variable region and light chain variable region (or variants thereof):

[0080] (i) the heavy chain variable region set forth in SEQ ID NO: 14, and the light chain variable region set forth in SEQ ID NO: 13; and

[0081] wherein the antigen binding domain that specifically binds to CD3 is selected from the group consisting of:

[0082] (1) ScFv shown in SEQ ID NO: 18,

[0083] (2) ScFv shown in SEQ ID NO: 19 or a variant thereof,

[0084] wherein the variant has 3, 2 or 1 amino acid differences with the corresponding variable region or ScFv, respectively, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity, respectively.

[0085] 9. The use according to any one of items 1 to 8, wherein the anti-EpCAM×CD3 bispecific antibody comprises

[0086] (1) A light chain-heavy chain pair that specifically binds to EpCAM, the light chain-heavy chain pair comprising a light chain and a heavy chain, or consisting thereof; wherein the light chain comprises a light chain variable region and a light chain constant region (preferably a sequence as shown in any one of SEQ ID NOs: 1 and 53-58), and the heavy chain comprises a heavy chain variable region, CH1 (preferably a sequence as shown in SEQ ID NO: 2), and a first Fc fragment; preferably, the first Fc fragment comprises a hinge region (preferably a sequence as shown in SEQ ID NO: 3), CH2 (preferably a sequence as shown in any one of SEQ ID NOs: 6, 7, 59-64), and CH3a;

[0087] (2) a fusion peptide that specifically binds to CD3, the fusion peptide comprising or consisting of a ScFv that specifically binds to CD3 and a second Fc fragment; preferably, the ScFv comprises, from N-terminus to C-terminus, a heavy chain variable region, a connecting peptide (preferably a sequence as shown in SEQ ID NO: 4), and a light chain variable region; the second Fc fragment comprises, from N-terminus to C-terminus, a hinge region (preferably a sequence as shown in SEQ ID NO: 3), CH2 (preferably a sequence as shown in any one of SEQ ID NOs: 6, 7, 59-64), and CH3b; preferably, the C-terminus of the light chain variable region is connected to the hinge region of the second Fc fragment via a connecting peptide (preferably a sequence as shown in SEQ ID NO: 5);

[0088] Preferably, the first Fc fragment and the second Fc fragment are human or humanized Fc fragments, such as human IgG Fc fragments, such as IgG1, IgG2, IgG3, IgG4, IgG5 Fc fragments;

[0089] Preferably, compared to the wild-type antibody, the first Fc fragment and / or the second Fc fragment comprises one or more substitutions that form a knob-and-hole pairing between the heavy chain and the fusion peptide, for example, T366 on one CH3 domain is replaced by a relatively large amino acid residue, such as tyrosine (Y) or tryptophan (W), and Y407 on the other CH3 domain is replaced by a relatively small amino acid residue, such as threonine (T), alanine (A) or valine (V), for example, comprising one or more substitutions listed in Table 10;

[0090] Preferably, the first Fc fragment and / or the second Fc fragment comprises one or more substitutions, 1) the substitutions form a salt bridge pairing between the heavy chain and the fusion peptide, for example, one CH3 domain comprises one or more substitutions, substituted with an amino acid residue that has a positive charge under physiological conditions, and the other CH3 domain comprises one or more substitutions, substituted with one or more amino acid residues that have a negative charge under physiological conditions, for example, the positively charged amino acid residue is arginine (R), histidine (H) or lysine (K), for example, the negatively charged amino acid residue is arginine (R), histidine (H) or lysine (K). The amino acid residue may be aspartic acid (D) or glutamic acid (E), for example, the substituted amino acid residues include one or more of D356, L368, K392, D399 and K409, such as one or more substitutions in Table 1, 2) the substitution forms a disulfide bond between the heavy chain and the fusion peptide, such as the substitutions in Table 2, and / or 3) the substitution results in a significant decrease in the binding ability between Fc and protein A, such as H435 and Y436 on a CH3 domain are replaced with arginine and phenylalanine, respectively, as shown in Table 11;

[0091] Preferably, wherein:

[0092] a) CH3b of the fusion peptide and CH3a of the heavy chain have a substitution pair that forms a knob-and-hole structure;

[0093] b) CH3b of the fusion peptide and CH3a of the heavy chain have a substitution pair that forms an ionic bond;

[0094] c) CH3b of the fusion peptide and CH3a of the heavy chain have a substitution pair that forms a disulfide bond; and / or

[0095] d) CH3b of the fusion peptide and CH3a of the heavy chain have substitutions that result in decreased binding to protein A;

[0096] Preferably, CH1 comprises the sequence of SEQ ID No: 2; and / or CL comprises a sequence selected from any one of SEQ ID Nos: 1, 53-58;

[0097] Preferably, the first Fc fragment and / or the second Fc fragment comprises a CH2 of any one of SEQ ID Nos: 6, 7, 59-64 and / or a CH3 of any one of SEQ ID Nos: 8, 9, 11, 12, 65-69;

[0098] Preferably, the sequences of CH3a and CH3b are selected from the group consisting of:

[0099] (1) one of the sequences is shown in SEQ ID NO:8, and the other sequence is shown in SEQ ID NO:11;

[0100] (2) one of the sequences is shown in SEQ ID NO: 9, and the other sequence is shown in SEQ ID NO: 12;

[0101] (3) one of the sequences is shown in SEQ ID NO:65, and the other sequence is shown in SEQ ID NO:67;

[0102] (4) one of the sequences is shown in SEQ ID NO: 9, and the other sequence is shown in SEQ ID NO: 68;

[0103] (5) one of the sequences is shown in SEQ ID NO:66, and the other sequence is shown in SEQ ID NO:69;

[0104] Preferably, the bispecific antibody is selected from the group consisting of:

[0105] (1) It comprises or consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises or consists of SEQ ID NO: 18, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 11; the heavy chain comprises or consists of SEQ ID NO: 14, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 8; and the light chain comprises or consists of SEQ ID NO: 13 and SEQ ID NO: 1;

[0106] (2) It comprises or consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises or consists of SEQ ID NO: 19, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 11; the heavy chain comprises or consists of SEQ ID NO: 14, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 8; and the light chain comprises or consists of SEQ ID NO: 13 and SEQ ID NO: 1;

[0107] (3) It comprises or consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises or consists of SEQ ID NO: 18, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 11; the heavy chain comprises or consists of SEQ ID NO: 16, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 8; and the light chain comprises or consists of SEQ ID NO: 15 and SEQ ID NO: 1;

[0108] (4) It comprises or consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises or consists of SEQ ID NO: 19, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 11; the heavy chain comprises or consists of SEQ ID NO: 16, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 8; and the light chain comprises or consists of SEQ ID NO: 15 and SEQ ID NO: 1;

[0109] (5) It comprises or consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises or consists of SEQ ID NO: 18, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO: 12; the heavy chain comprises or consists of SEQ ID NO: 14, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO: 9; and the light chain comprises or consists of SEQ ID NO: 13 and SEQ ID NO: 1;

[0110] (6) It comprises or consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises or consists of SEQ ID NO: 19, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO: 12; the heavy chain comprises or consists of SEQ ID NO: 14, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO: 9; and the light chain comprises or consists of SEQ ID NO: 13 and SEQ ID NO: 1;

[0111] (7) It comprises or consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises or consists of SEQ ID NO: 18, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 8; the heavy chain comprises or consists of SEQ ID NO: 14, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 11; and the light chain comprises or consists of SEQ ID NO: 13 and SEQ ID NO: 1.

[0112] (8) It comprises or consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises or consists of SEQ ID NO: 19, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 8; the heavy chain comprises or consists of SEQ ID NO: 14, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 11; and the light chain comprises or consists of SEQ ID NO: 13 and SEQ ID NO: 1;

[0113] (9) It comprises or consists of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises or consists of SEQ ID NO: 18, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 8; the heavy chain comprises or consists of SEQ ID NO: 16, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 11; and the light chain comprises or consists of SEQ ID NO: 15 and SEQ ID NO: 1.

[0114] (10) comprising or consisting of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises or consists of SEQ ID NO: 19, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 8; the heavy chain comprises or consists of SEQ ID NO: 16, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 11; and the light chain comprises or consists of SEQ ID NO: 15 and SEQ ID NO: 1.

[0115] (11) comprising or consisting of a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises or consists of SEQ ID NO: 18, SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO: 9; the heavy chain comprises or consists of SEQ ID NO: 14, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO: 12; and the light chain comprises or consists of SEQ ID NO: 13 and SEQ ID NO: 1;

[0116] (12) It comprises a fusion peptide, a heavy chain and a light chain, or consists of them; wherein the fusion peptide comprises SEQ ID NO:19, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:9, or consists of them; the heavy chain comprises SEQ ID NO:14, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:12, or consists of them; and the light chain comprises SEQ ID NO:13 and SEQ ID NO:1, or consists of them.

[0117] 10. The use according to any one of items 1 to 9, wherein the effective amount of the anti-EpCAM×CD3 bispecific antibody is: 10-50 μg or 50-400 μg, preferably the anti-EpCAM×CD3 bispecific antibody comprises a fusion peptide, a heavy chain and a light chain, or consists of them; wherein the fusion peptide comprises SEQ ID NO:18, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:11, or consists of them; the heavy chain comprises SEQ ID NO:14, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:8, or consists of them; the light chain comprises SEQ ID NO:13 and SEQ ID NO:1, or consists of them.

[0118] 11. The use according to any one of items 1 to 10, wherein the kit comprises two units, one unit comprising an effective amount of 10-50 μg / dose of the anti-EpCAM×CD3 bispecific antibody, and the other unit comprising an effective amount of 50-400 μg / dose of the anti-EpCAM×CD3 bispecific antibody.

[0119] 12. The use according to any one of items 1 to 11, wherein the anti-EpCAM×CD3 bispecific antibody comprises, or is composed of, a fusion peptide, a heavy chain and a light chain; wherein the fusion peptide comprises, or is composed of, SEQ ID NO:18, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:11; the heavy chain comprises, or is composed of, SEQ ID NO:14, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:8; and the light chain comprises, or is composed of, SEQ ID NO:13 and SEQ ID NO:1.

[0120] 13. A method for improving ascites in a patient, comprising the steps of administering an anti-EpCAM×CD3 bispecific antibody to the patient during an initial treatment period and a maintenance treatment period: wherein the initial treatment period comprises four administrations, the first administration dose is 10-50 μg (preferably 50 μg), the second, third, and fourth administration doses are 50-400 μg / time (preferably 400 μg / time) of the anti-EpCAM×CD3 bispecific antibody, and the maintenance treatment dose is 50-400 μg / time (preferably 400 μg / time) of the anti-EpCAM×CD3 bispecific antibody. Preferably, the patient is pretreated before the first and second administrations, and when the patient does not experience an allergic reaction and / or allergic reaction during or after subsequent administrations. or cytokine release syndrome, there is no need to pretreat the patient before subsequent administration. If the patient develops an allergic reaction and / or cytokine release syndrome during or after subsequent administration, the patient is pretreated before subsequent administration. The pretreatment is to administer antipyretic, analgesic and / or anti-allergic drugs (such as acetaminophen, dexamethasone sodium phosphate injection or diphenhydramine) to the patient, preferably intravenously, orally or intraperitoneally administering antipyretic, analgesic and / or anti-allergic drugs 30 minutes before administration (more preferably, the intraperitoneal infusion dose of dexamethasone is 5 mg, and the intraperitoneal infusion dose of diphenhydramine is 20 mg), wherein the anti-EpCAM×CD3 bispecific antibody is as defined in any one of items 6-11.

[0121] 14. The method of claim 13, wherein the initial treatment period comprises administering 50 μg, 400 μg, 400 μg, and 400 μg of the anti-EpCAM×CD3 bispecific antibody on day 1, day 4±2, day 11±3, and day 18±4, respectively, and preferably, the patient's ascites is drained (preferably fully drained) before the first and second doses, or the patient's ascites is drained (preferably fully drained) before each dose during the initial treatment period.

[0122] 15. The method described in any one of items 13-14, wherein in the initial treatment period, before the administration of the anti-EpCAM×CD3 bispecific antibody, the patient's peritoneal cavity is perfused with normal saline (preferably 100-650 ml), and after the administration of the anti-EpCAM×CD3 bispecific antibody (preferably the anti-EpCAM×CD3 bispecific antibody is dissolved in 50-250 ml of normal saline), the patient's peritoneal cavity is perfused with normal saline (preferably 0-250 ml).

[0123] 16. The method described in any one of items 13-15, wherein in the initial treatment period, before administering the anti-EpCAM×CD3 bispecific antibody, 500 ml of normal saline is perfused into the patient's peritoneal cavity, and after administering the anti-EpCAM×CD3 bispecific antibody, normal saline (preferably 0-250 ml) is perfused into the patient's peritoneal cavity, wherein the anti-EpCAM×CD3 bispecific antibody is a solution of 50-400 μg (preferably 50 μg) of a lyophilized preparation dissolved in 100-250 ml (preferably 250 ml) of normal saline, and the administration time of the anti-EpCAM×CD3 bispecific antibody is preferably 0.5-3 hours.

[0124] 17. The method described in any one of items 13-16, wherein the maintenance treatment period comprises administering the anti-EpCAM×CD3 bispecific antibody once every 1 or 2 weeks after 18 days of administration, preferably wherein the anti-EpCAM×CD3 bispecific antibody is a solution of 400 μg of a lyophilized preparation dissolved in 250 ml of normal saline, and preferably the administration time of the anti-EpCAM×CD3 bispecific antibody is 0.5-3 hours.

[0125] 18. The method according to any one of items 13 to 17, wherein 5 mg of dexamethasone sodium phosphate injection is administered intramuscularly, intravenously, or intraperitoneally 30 minutes before the first and second doses of the drug during the initial treatment period; or 20 mg of diphenhydramine injection is administered intramuscularly or intraperitoneally; if no infusion reaction occurs during the period, pretreatment can be stopped for the third and subsequent doses.

[0126] The initial treatment period of the anti-EpCAM×CD3 bispecific antibody: 50 μg is administered by intraperitoneal perfusion for the first treatment, followed by intraperitoneal perfusion on day 4 or day 4±2, day 11 or day 11±3, and day 18 or day 18±4, 400 μg each time. Before each administration, the ascites is fully drained, and 500 ml of normal saline and the anti-EpCAM×CD3 bispecific antibody diluted with 250 ml of normal saline are sequentially perfused. After the end of the administration, 0-250 ml of normal saline is administered by intraperitoneal perfusion.

[0127] Maintenance treatment period of the anti-EpCAM×CD3 bispecific antibody: After treatment on day 18 or day 18±4, 400 μg is administered once every 7 days or every 14 days. During the maintenance treatment period, ascites drainage is no longer performed before administration, and normal saline intraperitoneal perfusion is no longer performed before and after administration.

[0128] Preferably, the intraperitoneal infusion time of the anti-EpCAM×CD3 bispecific antibody is 0.5-3 hours.

[0129] In this article, "infusion reaction" refers to allergic reactions, such as adverse reactions caused by complement activation, autoreactive lymphocyte activation and massive release of inflammatory factors caused by the molecular structure of monoclonal antibodies ( S,BLASCO I, FABREGAT L, et al. Management of infusion reactions to systemic anticancer therapy: ESMO clinical practice guidelines[J]. Ann Oncol, 2017, 28(suppl_4):iv100.).

[0130] In this article, "cytokine release syndrome" refers to a clinical syndrome that is seriously life-threatening and is caused by the overactivation of various immune cells such as T cells, macrophages, and NK cells, which is induced by multiple factors and characterized by a rapid increase in the levels of inflammatory mediators such as interleukins, interferons, and chemokines. It is a common adverse reaction of immunomodulatory drugs and is common in patients with malignant tumors, infectious diseases, autoimmune diseases, and monogenic diseases (Erratum: LEE DW, GARDNER R, PORTER DL, et al. Current concepts in the diagnosis and management of cytokine release syndrome [J]. Blood, 2014, 124(2): 188-195).

[0131] In this article, "antipyretic and analgesic drugs" generally refer to drugs that have the effects of suppressing immunity, and / or inhibiting prostaglandin synthesis, and / or inhibiting inflammatory responses, and have antipyretic, and / or analgesic, and / or anti-allergic effects, such as antihistamines, glucocorticoids and other steroidal and non-steroidal drugs.

[0132] The form suitable for intraperitoneal perfusion refers to a pharmaceutical preparation that meets the requirements of an injection.

[0133] In this article, Day 1 of the initial treatment period refers to the day when the patient first receives this product.

[0134] In this article, "drainage of ascites" refers to the removal of ascites by placement of a drainage tube via peritoneal puncture.

[0135] In this article, drainage is preferably adequate drainage, which means that the ascites cannot be drained and the maximum depth of ascites in the supine position on B-ultrasound is ≤3cm; or the drainage volume within 1 hour is ≤100ml; the above situations need to exclude ascites separation or drainage tube blockage.

[0136] In the present invention, amino acid substitutions in CH3 are shown in Tables 1 and 2.

[0137] Table 1. Combinations of CH3 amino acid substitutions that form ionic bonds between monovalent units and single-chain units to improve heterodimer pairing efficiency.

[0138] Table 2. Combinations of CH3 amino acid substitutions to form disulfide bonds between monovalent units and single-chain units to improve heterodimer pairing efficiency

[0139] Table 10. Combinations of CH3 amino acid substitutions form knob-in-hole pairs between monovalent units and single-chain units to improve heterodimer pairing efficiency.

[0140] Table 11. A CH3 amino acid substitution results in decreased binding to Protein A.

[0141] In the present invention, the preparation of the anti-EpCAM×CD3 bispecific antibody is as described in WO2023 / 087255, which is incorporated into the present invention by reference. The specific sequence of the EpCAM×CD3 bispecific antibody of the present invention is shown in Table 3.

[0142] Table 3. Amino acid sequence information corresponding to each antibody molecule

[0143] The anti-EpCAM x CD3 bispecific antibody described in this invention is a recombinant bispecific antibody, using the human IgG1 constant region as its structural framework and primarily composed of an anti-EpCAM heavy chain, an anti-EpCAM light chain, and an anti-CD3 single chain. Compared to the all-mouse antibody Catumaxomab, produced using earlier hybrid-hybridoma technology, it offers the following advantages in structural design, production, and quality:

[0144] 1) The anti-EpCAM×CD3 bispecific antibody is a human-mouse chimeric antibody with a higher degree of humanization, which is beneficial for reducing the risk of immunotoxicity;

[0145] 2) The anti-EpCAM × CD3 bispecific antibody utilizes current mainstream monoclonal antibody production and testing technology, has a stable production process, and has a lower proportion of multimeric impurities, a more stable structure, and a 24-month shelf life (the published shelf life of Catumaxol is 18 months).

[0146] 3) The anti-EpCAM×CD3 bispecific antibody has relatively weakened affinity for CD3 and EpCAM. This weakening does not affect T cell activation and targeting of tumor cells, but can reduce nonspecific activation and overactivation of T cells, and reduce the risk of damage to normal tissues with low EpCAM expression, thereby reducing off-target toxicity and AEs related to cytokine release symptoms.

[0147] The volume of intraperitoneal fluid effusion varies significantly between patients, generally ranging from 1 to 7 L. This fluid contains numerous viable or apoptotic tumor, immune cells, tissue cells, and inflammatory factors, which can promote exudate exudation and block lymphatic drainage. Inflammatory factors also contain immunosuppressive factors. Therefore, intraperitoneal drug administration typically involves first draining the ascites thoroughly, followed by reinfusion of a defined volume of saline and medication. This helps control the drug concentration range and promotes uniform distribution, allowing the immune-activating and tumor-targeting anti-EpCAM×CD3 bispecific antibody to exert its pharmacological effects. However, frequent drainage can lead to significant nutrient loss and electrolyte imbalances, increasing the risk of infection and accelerating disease progression, leading to worsening symptoms of malignant ascites and even death. Therefore, different intraperitoneal drug administration methods can result in varying therapeutic outcomes.

[0148] Therefore, in the present invention, after draining the ascites and before administering the anti-EpCAM×CD3 bispecific antibody, normal saline is perfused into the patient's peritoneal cavity, which is beneficial for controlling the concentration range of the drug, uniformly dispersing the drug in the peritoneal cavity, and improving the immunosuppressive environment in the peritoneal cavity. At the same time, after administration, according to the patient's tolerance, an appropriate dose of normal saline is perfused into the patient's peritoneal cavity to further promote uniform dispersion of the drug in the peritoneal cavity, thereby avoiding an increase in adverse reactions due to excessive local drug concentration.

[0149] In the present invention, the objective response rate of ascites refers to the number of patients with ascites reduced by more than 50% on the 28th day after treatment / the number of treated patients; the disease control rate of ascites refers to the number of patients with ascites reduced by more than 20% on the 28th day after treatment / the number of treated patients; and the overall survival (OS) refers to the time from the start of randomization to the death of the patient due to any cause.

[0150] The molecular design advantages of the anti-EpCAM×CD3 bispecific antibody described in the present invention, such as its higher degree of humanization and weakened affinity compared with catumaxol, have been preliminarily verified in a Phase II clinical study. It has comparable efficacy and better safety in patients with tumor progression after second-line treatment and malignant ascites (ovarian cancer is platinum-resistant).

[0151] The mechanism of MA generation is generally considered to be:

[0152] 1) Tumor cells block lymphatic vessels and hinder lymphatic drainage, resulting in reduced outflow of peritoneal exudate;

[0153] 2) Tumor invasion of the peritoneum leads to damage to the vascular endothelium and increased exudate; or

[0154] 3) Tumor cells produce a variety of inflammatory factors (such as interleukin-6 [IL-6]) or cell growth factors (such as vascular endothelial growth factor [VEGF]), which lead to tumor angiogenesis and increased capillary permeability in the tumor and peritoneum.

[0155] In this article, malignant ascites is caused by malignant tumors, such as epithelial malignancies. As a serious symptom caused by malignant tumors, the treatment goals of malignant ascites are to control ascites symptoms and improve the patient's quality of life.

[0156] For patients, controlling ascites reaccumulation and reducing the number of punctures / drainages significantly improves their quality of life. Therefore, compared to evaluating tumor response, controlling malignant ascites is often evaluated by puncture-free survival (PuFS), the time from the end of treatment to the next need for puncture / drainage, as a measure of clinical benefit for ascites. BRIEF DESCRIPTION OF THE DRAWINGS

[0157] FIG1 is a schematic diagram of an exemplary structure of an anti-EpCAM×CD3 bispecific antibody, wherein EpCAM represents epithelial cell adhesion molecule; CD3 represents human leukocyte cluster of differentiation 3. DETAILED DESCRIPTION

[0158] The following describes the method and application of the present invention in conjunction with the accompanying drawings. The examples given are only for the purpose of explaining the present invention and are not intended to limit the scope of the present invention. For those skilled in the art of the present invention, simple modifications or substitutions can be made without departing from the concept of the present invention, and all of these should be considered to fall within the scope of protection of the present invention.

[0159] Example 1. Preparation of anti-EpCAM×CD3 bispecific antibody

[0160] Prepared according to the method of WO2023 / 087255. The bispecific antibody structure for EpCAM and CD3 includes (a) a light chain-heavy chain pair that specifically binds to EpCAM, wherein the light chain is bound to the heavy chain through a disulfide bond; and (b) a fusion peptide that specifically binds to CD3, wherein the fusion peptide comprises a single-chain variable fragment (scFv) and an Fc fragment having a CH2 domain and a CH3 domain, and the fusion peptide is bound to the heavy chain through a disulfide bond. The bispecific antibody forms a YBODY structure as described in Figure 1, wherein the anti-CD3 VL is connected to the hinge region and CH2 through a linker, and the anti-EpCAM heavy chain Fc and the anti-CD3 fusion peptide (with human IgG heavy chain Fc as the skeleton) Fc are subjected to amino acid mutation modification so that each of them does not form a homodimer, but is easy to form a heterodimer.

[0161] Using existing plasmids or synthetic gene fragments as templates, each chain of the bispecific antibody was amplified by PCR and overlapping PCR. Each antibody chain was then cloned into the pcDNA3.1 vector (Invitrogen) by enzyme ligation or recombination. The detailed sequence information for each antibody chain is shown in Table 3 and the sequence listing.

[0162] According to conventional methods, a lyophilized powder of a bispecific antibody against EpCAM×CD3 was prepared.

[0163] Example 2. Phase II controlled clinical study of patients with malignant ascites receiving injection of recombinant anti-EpCAM×CD3 bispecific antibody or puncture drainage

[0164] Taking M701A as an example, the effect of an anti-EpCAM and CD3 bispecific antibody on improving ascites was studied. In the bispecific antibody, the anti-EpCAM-specific light chain comprises SEQ ID NO:13 and SEQ ID NO:1 (or the full-length sequence set forth in SEQ ID NO:70), the anti-EpCAM-specific heavy chain comprises SEQ ID NO:14, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6, and SEQ ID NO:8 (or the full-length sequence set forth in SEQ ID NO:71); and the anti-CD3-specific fusion peptide comprises SEQ ID NO:18, SEQ ID NO:5, SEQ ID NO:3, SEQ ID NO:6, and SEQ ID NO:11 (or the full-length sequence set forth in SEQ ID NO:72). The prepared injection solution may comprise 0.5 mg / mL of the anti-EpCAM and CD3 bispecific antibody, 30 mM histidine and histidine hydrochloride buffer, 5.0% trehalose, 0.05% PS80, and a pH value of 5.2. The inventors have demonstrated that the injection can inhibit the occurrence of physical and chemical changes to the greatest extent, thereby improving product quality, stability, and safety for clinical use. After reconstitution, the injection is suitable for intravenous, subcutaneous, intramuscular, thoracic, or intraperitoneal administration and can be used as a drug for clinical use.

[0165] Experimental Design:

[0166] After the patients signed the informed consent, they were screened before joining the trial.

[0167] Inclusion criteria:

[0168] Exclusion criteria:

[0169] Eligible patients were randomly assigned to either the M701A treatment group or the puncture drainage group. Starting from day 18 (D18) after enrollment, puncture-free survival (PFS) and median survival (MSS) were compared between the M701A and puncture drainage groups. PFS was defined as the time from the completion of drainage before the fourth dose on D18 of the initial treatment period to the start of the next drainage (calculated by the time of drainage) or death, whichever occurred first. Median values ​​and 95% confidence intervals for PuFS and OS were estimated using the Kaplan-Meier method, and the log-rank test was used for comparison between the two groups.

[0170] The M701A treatment group (M701A) was divided into two treatment regimen subgroups, hereinafter referred to as M701A1 and M701A2. The treatment regimen was carried out under the guidance of physicians with experience in tumor treatment.

[0171] Treatment plan for M701A1 group:

[0172] 1) Initial treatment period: 50 μg was administered intraperitoneally on the first day of treatment (day 1), followed by 400 μg intraperitoneally on days 4±2, 11±3, and 18±4. Before the first and second doses, the ascites was fully drained and perfused sequentially with 500 ml of normal saline and M701A diluted with 250 ml of normal saline. After the end of the dosing period, 0-250 ml (adjusted by the investigator based on the patient's abdominal tolerance) of normal saline was administered intraperitoneally.

[0173] 2) Maintenance treatment period: After the 18th day of treatment, 400 μg of M701A was administered once a week (7 days). During the maintenance treatment period, ascites drainage was not performed before administration. During the maintenance treatment period, only 250 ml of M701A diluted with normal saline was intraperitoneally infused, and no normal saline was administered before or after administration.

[0174] 3) Pretreatment: Administer 5 mg of dexamethasone sodium phosphate injection intramuscularly, intravenously, or intraperitoneally, or 20 mg of diphenhydramine injection intramuscularly or intraperitoneally 30 minutes before the first and second doses of the initial treatment period. If no infusion reaction and / or cytokine release syndrome occurs during this period, pretreatment can be discontinued before the third and subsequent doses.

[0175] Treatment plan for group M701A2:

[0176] 1) Initial Treatment Period: 50 μg was administered intraperitoneally for the first treatment (Day 1), followed by 400 μg of M701A per dose on Days 4±2, 11±3, and 18±4. Before each dose, the ascites was fully drained and perfused sequentially with 500 ml of normal saline and then M701A diluted in 250 ml of normal saline. Following the completion of the dosing period, 0-250 ml of normal saline (adjusted by the investigator based on the patient's intraperitoneal tolerance) was administered intraperitoneally.

[0177] 2) Maintenance Treatment Period: After treatment day 18, 400 μg of M701A was administered every 2 weeks (14 days). Ascites drainage was not performed before dosing during the maintenance treatment period. Only 250 ml of M701A diluted in normal saline was administered intraperitoneally during the maintenance treatment period. No normal saline was administered intraperitoneally before or after dosing.

[0178] 3) Pretreatment: Administer 5 mg of dexamethasone sodium phosphate injection intramuscularly, intravenously, or intraperitoneally, or 20 mg of diphenhydramine injection intramuscularly or intraperitoneally 30 minutes before the first and second doses of the initial treatment period. If no infusion reaction and / or cytokine release syndrome occurs during this period, pretreatment can be discontinued before the third and subsequent doses.

[0179] Treatment plan for the puncture and drainage control group (Control):

[0180] Patients underwent puncture drainage on D1 and D18, and puncture drainage was performed on D2-D17 according to the patient's needs.

[0181] Test results:

[0182] At approximately 5 months of follow-up (mid-term) and at the end of follow-up (end-term), PuFS (Puncture-Free Survival) was analyzed for 57 patients in the M701A group (14 in the M701A1 group and 43 in the M701A2 group) and 40 patients in the control group. Overall Survival (OS) was also analyzed for 57 patients in the M701A group (14 in the M701A1 group and 50 in the M701A2 group (including 7 patients who crossed over after progression in the control group)) and 40 patients in the control group. The interim analysis results are detailed in Tables 4 and 5 below, and the end-of-term analysis results are detailed in Tables 6 and 7 below. The results showed that the M701A2 group had a longer-lasting PuFS and OS than the M701A1 and control groups, while the M701A1 group had PuFS and OS comparable to those in the control group.

[0183] Table 4: PuFS results of each group at a median follow-up of approximately 5 months

[0184] Note: HR (95% CI) = 0.39 (0.21, 0.72) compared with the control group, P value = 0.0015.

[0185] Table 5: OS results of each group at a median follow-up of approximately 5 months

[0186] Note: HR (95% CI) = 0.65 (0.37, 1.16) compared with the control group, P value = 0.1419.

[0187] Table 6: PuFS results of each group at the end of follow-up

[0188] Note: HR (95% CI) = 0.43 (0.22, 0.81) compared with the control group, P value = 0.0065.

[0189] Table 7: OS results of each group at the end of follow-up

[0190] Note: HR (95% CI) = 0.77 (0.46, 1.26) compared with the control group, P value = 0.2946.

[0191] In addition, the M701A2 group showed good safety, with the adverse event rate of ≥CTCA grade 3 being 58%.

[0192] Because both M701A and Catumaxomab (an anti-EpCAM × CD3 bispecific antibody approved by the European Medicines Agency (EMA)) utilize both immune and targeted therapeutic mechanisms, targeting EpCAM on tumor cells and CD3 on immune cells, and involving the biological mechanisms of immune and T cell activation, cytokine release syndrome (CRS) or CR-related symptoms are of particular concern for these drugs. Among CR-related symptoms, fever (61.8% for Catumaxomab and 18.0% for M701A), nausea (45.2% for Catumaxomab and 12.0% for M701A), vomiting (37.6% for Catumaxomab and 24.0% for M701A), and tachycardia (15.3% for Catumaxomab and 14.0% for M701A) were significantly less frequent with M701A2 than with Catumaxomab (see Tables 8 and 9 for details).

[0193] Table 8. Adverse Events with ≥1% Cytokine Release Composite-Related Symptoms

[0194] Table 9. Adverse events (CTCAE grade)

[0195] Notes to Tables 8 and 9: Catumaxol data are derived from the Phase II / III clinical study, IP-REM-AC-01. Catumaxol dosing regimen: Each patient is limited to four doses; prior to each dose, ascites must be fully drained and 1000 mg of paracetamol, an antipyretic and analgesic, must be administered. Prior to each dose, 500 mL of normal saline solution must be intraperitoneally instilled. On days 0, 3, 7, and 10, 10, 20, 50, and 150 μg of catumaxol diluted in 250 ml of normal saline solution must be administered intraperitoneally, respectively. The first dose should be administered within 6 hours, and the second, third, and fourth doses should be administered within no less than 3 hours.

[0196] Example 3. Efficacy and safety of intraperitoneal perfusion of recombinant anti-EpCAM and CD3 bispecific antibodies versus peritoneal puncture and drainage in patients with malignant ascites caused by advanced epithelial solid tumors

[0197] The main objective of this study is to evaluate the effectiveness of intraperitoneal perfusion injection of recombinant anti-EpCAM and CD3 bispecific antibody (experimental group) compared with peritoneal puncture and drainage (control group) in patients with malignant ascites caused by advanced epithelial solid tumors. The primary endpoint is puncture-free survival (PuFS). Secondary endpoints are overall survival (OS), time to next puncture and drainage, patient-reported outcome score, 1- and 2-month PuFS rates, 6-month survival rate, adverse events, and pharmacokinetics.

[0198] The experimental group adopted the treatment regimen of the M701A2 group in Example 2, and the control group adopted abdominal puncture and drainage. This study verified the good treatment effect and safety of the experimental group through a large sample size.

[0199] Sequence Listing

Claims

1. A method for improving or treating malignant ascites in a subject, the method comprising: 1) in the initial treatment period, administering the anti-EpCAM×CD3 bispecific antibody four times, wherein the first administration dose is about 10 to 50 μg, preferably about 50 μg, and the second, third, and fourth administration doses are about 50 to 400 μg, preferably about 400 μg each, and the subject undergoes ascites drainage before each administration, and; 2) during the maintenance treatment period, administering the anti-EpCAM×CD3 bispecific antibody once every 2 weeks, with each dose ranging from about 50 to 400 μg, and preferably about 400 μg; The anti-EpCAM×CD3 bispecific antibody is in a form suitable for intraperitoneal perfusion.

2. An anti-EpCAM×CD3 bispecific antibody for improving or treating malignant ascites in a subject, wherein the method of improvement or treatment comprises: 1) in the initial treatment period, administering the anti-EpCAM×CD3 bispecific antibody four times, wherein the first administration dose is about 10 to 50 μg, preferably about 50 μg, and the second, third, and fourth administration doses are about 50 to 400 μg, preferably about 400 μg each, and the subject undergoes ascites drainage before each administration, and; 2) during the maintenance treatment period, administering the anti-EpCAM×CD3 bispecific antibody once every 2 weeks, with each dose ranging from about 50 to 400 μg, and preferably about 400 μg; The anti-EpCAM×CD3 bispecific antibody is in a form suitable for intraperitoneal perfusion.

3. The method according to claim 1 or the anti-EpCAM×CD3 bispecific antibody according to claim 2, wherein the anti-EpCAM×CD3 bispecific antibody comprises (a) a light chain-heavy chain pair that specifically binds to EpCAM, wherein the light chain is bound to the heavy chain through a disulfide bond; and (b) a fusion peptide that specifically binds to CD3, wherein the fusion peptide comprises a single-chain variable fragment (scFv) and an Fc fragment having a CH2 domain and a CH3 domain, and the fusion peptide is bound to the heavy chain through a disulfide bond.

4. The method or anti-EpCAM×CD3 bispecific antibody according to any one of claims 1 to 3, wherein Before the first and second administrations of the bispecific antibody during the initial treatment period, the subject is pretreated by administering an antipyretic analgesic. Thereafter, if the subject does not experience an infusion reaction and / or cytokine release syndrome during or after the administration of the bispecific antibody, there is no need to administer an antipyretic analgesic before the administration of the bispecific antibody. If the subject experiences an infusion reaction and / or cytokine release syndrome during or after the administration of the bispecific antibody, an antipyretic analgesic is administered before the administration of the bispecific antibody. 5 . The method or anti-EpCAM×CD3 bispecific antibody according to claim 4 , wherein the analgesic and antipyretic drug is an antihistamine, a steroidal and / or nonsteroidal drug, such as a glucocorticoid, for example dexamethasone, diphenhydramine or acetaminophen. 6 . The method or anti-EpCAM×CD3 bispecific antibody according to claim 5 , wherein the pretreatment is intraperitoneal perfusion of about 5 mg of dexamethasone or about 20 mg of diphenhydramine.

7. The method or anti-EpCAM×CD3 bispecific antibody according to any one of the preceding claims, wherein In the initial treatment period, the four administrations are respectively on day 1, day 4 or day 4±2, day 11 or day 11±3 and day 18 or day 18±4, preferably at a dose of about 50 μg, 400 μg, 400 μg, 400 μg, respectively.

8. The method or anti-EpCAM×CD3 bispecific antibody according to any one of the preceding claims, wherein After the drainage is completed and before the antibody is administered, the subject is intraperitoneally perfused with saline, preferably about 100 to 650 ml of saline. After the antibody is administered, the subject is intraperitoneally perfused with saline, preferably about 0 to 250 ml of saline.

9. The method or anti-EpCAM×CD3 bispecific antibody according to any one of the preceding claims, wherein The anti-EpCAM×CD3 bispecific antibody is a solution containing 0.01 to 5 mg / mL, preferably 0.5 mg / mL, anti-EpCAM×CD3 bispecific antibody, 30 mM histidine and histidine hydrochloride buffer, 5.0% (w / v) trehalose and 0.05% (w / v) PS80, and having a pH of 5.

2.

10. The method or anti-EpCAM x CD3 bispecific antibody according to any one of the preceding claims, wherein The heavy chain-light chain pair that specifically binds to EpCAM comprises: (i) CDRH1, CDRH2, and CDRH3 contained in the heavy chain variable region shown in SEQ ID NO: 14, and (ii) CDRL1, CDRL2, and CDRL3 contained in the light chain variable region shown in SEQ ID NO: 13, Preferably, the sequence of CDRL1 is shown in SEQ ID NO: 25, the sequence of CDRL2 is shown in SEQ ID NO: 26, the sequence of CDRL3 is shown in SEQ ID NO: 27, the sequence of CDRH1 is shown in SEQ ID NO: 28, the sequence of CDRH2 is shown in SEQ ID NO: 29, and the sequence of CDRH3 is shown in SEQ ID NO: 30; or (iii) CDRH1, CDRH2, and CDRH3 contained in the heavy chain variable region shown in SEQ ID NO: 16, and (iv) CDRL1, CDRL2, and CDRL3 contained in the light chain variable region shown in SEQ ID NO: 15, Preferably, the sequence of CDRL1 is shown as SEQ ID NO:31, the sequence of CDRL2 is shown as SEQ ID NO:32, the sequence of CDRL3 is shown as SEQ ID NO:33, the sequence of CDRH1 is shown as SEQ ID NO:34, the sequence of CDRH2 is shown as SEQ ID NO:35, and the sequence of CDRH3 is shown as SEQ ID NO:36; The fusion peptide that specifically binds to CD3 comprises: (i) CDRH1, CDRH2, and CDRH3 contained in the heavy chain variable region set forth in SEQ ID NO:43, and CDRL1, CDRL2, and CDRL3 contained in the light chain variable region set forth in SEQ ID NO:44, Preferably, the sequence of CDRH1 is shown in SEQ ID NO:37, the sequence of CDRH2 is shown in SEQ ID NO:38, and the sequence of CDRH3 is shown in SEQ ID NO:39, the sequence of CDRL1 is shown in SEQ ID NO:40, the sequence of CDRL2 is shown in SEQ ID NO:41, and the sequence of CDRL3 is shown in SEQ ID NO:42; or (ii) CDRH1, CDRH2, and CDRH3 contained in the heavy chain variable region set forth in SEQ ID NO:51, and CDRL1, CDRL2, and CDRL3 contained in the light chain variable region set forth in SEQ ID NO:52, Preferably, the sequence of CDRH1 is shown as SEQ ID NO:45, the sequence of CDRH2 is shown as SEQ ID NO:46, and the sequence of CDRH3 is shown as SEQ ID NO:47, the sequence of CDRL1 is shown as SEQ ID NO:48, the sequence of CDRL2 is shown as SEQ ID NO:49, and the sequence of CDRL3 is shown as SEQ ID NO:

50.

11. The method or anti-EpCAM×CD3 bispecific antibody according to any one of the preceding claims, wherein The heavy chain-light chain pair that specifically binds to EpCAM comprises: (i) a heavy chain variable region having a sequence as set forth in SEQ ID NO: 14, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical thereto, and a light chain variable region having a sequence as set forth in SEQ ID NO: 13, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical thereto; or (ii) a heavy chain variable region having a sequence as set forth in SEQ ID NO: 16, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical thereto, and a light chain variable region having a sequence as set forth in SEQ ID NO: 15, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical thereto; and The fusion peptide that specifically binds to CD3 comprises: (i) a heavy chain variable region having a sequence as set forth in SEQ ID NO:43, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and a light chain variable region having a sequence as set forth in SEQ ID NO:44, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, or (ii) a heavy chain variable region having a sequence as set forth in SEQ ID NO:51, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and a light chain variable region having a sequence as set forth in SEQ ID NO:52, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; Preferably, the anti-EpCAM×CD3 bispecific antibody comprises a heavy chain variable region having the sequence of SEQ ID NO: 14, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; a light chain variable region having the sequence of SEQ ID NO: 13, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; and a fusion peptide that specifically binds to CD3 comprising the sequence of SEQ ID NO: 18 or SEQ ID NO: 19, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

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