WO2024082269A1 - Application of bispecific antibody in immune cell therapy - Google Patents

Abstract

Provided are an application of a bispecific antibody in immune cell therapy, and a method for improving immune cell killing activity.

Description

Application of bispecific antibodies in immune cell therapy Technical Field

The present invention belongs to the field of immunotherapy. Specifically, the present invention relates to the application of bispecific antibodies in immune cell therapy and a method for improving the killing activity of immune cells.

Background technique

Cell therapy includes CAR-T cell therapy, tumor infiltrating lymphocyte therapy (TIL), NK cell therapy, DC cell therapy, CIK cell therapy, stem cells and other cell drugs. Cell therapy drugs are taken from human samples, including blood samples and tissue samples, and the sampling sites include peripheral blood, tumor in situ, paracancerous tissue, lymph nodes, umbilical cord, bone marrow and other sites. TIL, CIK, NK and other cells that have not undergone cell engineering are often used in tumor treatment. Through in vitro culture, the number and anti-tumor activity of cells can be increased. For patients with advanced cancer or patients who can no longer be treated by surgery, radiotherapy and chemotherapy, the reinfusion of such in vitro cultured cells will have a better therapeutic effect and better safety. However, such cells that have not undergone cell engineering are not tumor-targeted, and a large number of reinfused immune cells cannot be effectively enriched in the tumor site, which greatly reduces the efficacy. Genetic engineering of cells will introduce some safety risks, and the cost is high, which has limitations in application.

TIL refers to tumor-infiltrating lymphocytes, which are obtained by isolating, culturing and activating lymphocytes in tumor tissue in vitro, relieving the inhibition of the tumor microenvironment, reactivating lymphocytes and regaining their ability to kill tumor cells. Generally, more than 90% of TIL is composed of CD3+ cell groups, because they have been stimulated by a large number of tumor-related antigens and have a relatively rich T cell receptor (T cell receptor, TCR) on the cell surface. When the number of TIL cells reaches the order of one billion to tens of billions, they are infused back into the patient's body. Under the stimulation of tumor cells in the body, TIL cells once again exert their anti-tumor function, killing or inhibiting the proliferation of tumor cells.

Bispecific antibodies (BsAbs) are considered to be effective candidate therapeutic drugs for the treatment of cancer. There are many forms of bispecific antibodies, one of which is bispecific T-cell engagers (Bite), which can usually target tumor cell surface antigens and immune cell surface antigens at the same time, and guide and activate T lymphocytes to target and kill tumor cells through dual-target binding, playing a significant role in cancer. However, tumor patients often have weak immune function, and have the disadvantages of small number and weak activity of immune cells in the body, which seriously reduce the anti-tumor effect of Bite.

The M701 series antibodies are bispecific antibodies against EpCAM and CD3, among which EpCAM (Recombinant Epithelial Cell Adhesion Molecule) is a highly expressed antigen on the surface of tumor cells of epithelial cell-derived tumors. Because the vast majority of tumors are of epithelial cell origin, such as colorectal cancer, gastric cancer, breast cancer, ovarian cancer, lung cancer (such as non-small cell lung cancer), prostate cancer, pancreatic cancer, liver cancer, retinoblastoma, esophageal cancer, kidney cancer, renal clear cell tumor, skin squamous cell carcinoma, skin basal cell carcinoma, sarcoma, nasal glioma, craniopharyngioma, thyroid cancer, cholangiocytoma, bladder cancer, head and neck tumors, cervical cancer, or oral cancer, etc., EpCAM was first used as a tumor marker and later as a target for tumor treatment; CD3 is a surface marker of T cells in lymphocytes and the first activation signal of T cells. When the M701 series antibodies bind to EpCAM on tumor cells and CD3 on T cells at the same time, T cells will be activated and have an immune killing effect on EpCAM-positive cells.

Therefore, by incubating Bite (such as the anti-EpCAM and CD3 bispecific T cell engager M701 series antibodies) with immune cells (such as TIL) in vitro, an antibody-cell complex can be formed. After the antibody-cell complex that has proliferated in large quantities in vitro is returned to the patient's body, the binding force between the M701 series antibodies and the tumor cell surface antigen EpCAM can be used to effectively increase the targeting of the antibody-cell complex to tumor cells, allowing more TIL cells to be enriched in the tumor site, promoting the binding between TIL cells and tumor cells, and activating the anti-tumor activity of TIL cells.

Summary of the invention

On the one hand, the present invention provides a bispecific antibody and an immune cell complex, wherein the bispecific antibody is an anti-EpCAM and CD3 bispecific antibody, and the immune cell binds to the anti-EpCAM and CD3 bispecific antibody. Preferably, the immune cell is selected from TIL cells, CIK cells, LAK cells, T cells, and NKT cells.

On the other hand, the present invention relates to a kit comprising immune cells and anti-EpCAM and CD3 bispecific antibodies, or comprising the bispecific antibody and immune cell complex described in the present invention. Preferably, the kit also comprises drugs (such as small molecule drugs or macromolecule drugs) for treating tumors and/or malignant ascites, malignant effusion, malignant pleural effusion, etc.

In another aspect, the present invention relates to a method for improving the killing activity of anti-EpCAM and CD3 bispecific antibodies and/or immune cells, the method comprising combining the immune cells with anti-EpCAM and CD3 bispecific antibodies, preferably culturing the immune cells with anti-EpCAM and CD3 bispecific antibodies.

In another aspect, the present invention relates to a pharmaceutical composition comprising the bispecific antibody and immune cell complex of the present invention and a pharmaceutically acceptable carrier.

In another aspect, the present invention relates to a method for treating tumors and/or malignant ascites, malignant effusion, malignant pleural effusion, etc., comprising administering a therapeutically effective amount of the bispecific antibody and immune cell complex or pharmaceutical composition of the present invention to a subject.

In some embodiments, the immune cells are TIL cells.

In some embodiments, the TIL cells are from solid tumor tissue samples such as non-small cell lung cancer, breast cancer, cervical cancer, colorectal cancer, liver cancer, melanoma, gastric cancer, liver cancer, or ovarian cancer.

In some embodiments, the TIL cells are prepared by the following steps: solid tumor tissue samples are digested using collagenase digestion solution in a 37°C shaker for 1 hour, the supernatant is removed after centrifugation, the cell pellet is resuspended in X-VIVO15 culture medium containing 1000-6000 IU/ml IL-2, inoculated into a 24-well plate, and placed in a carbon dioxide incubator for static culture, and fresh culture medium is replaced every 1-2 days.

In some embodiments, the preparation further comprises the following steps: adding 1000-6000 IU/ml IL-2, 10-100 ng/ml CD3 antibody and 10-20×10 ^{6 cells/ml irradiated (50-100 Gray) PBMC cells per 1-2×10 6 cells} /ml, mixing and placing in a carbon dioxide incubator for culture; thereafter, adding an appropriate volume of X-VIVO15 culture medium containing 1000-6000 IU/ml IL-2 every 2-3 days. Preferably, during the culture process, 1-5% AB serum (Gemini, 100-512) and immune serum substitute (Gibco, A25961) can be added according to the cell state, and a sufficient number of TIL cells can be harvested after the cells are cultured for about 15 days.

In some embodiments, the anti-EpCAM and CD3 bispecific antibody is selected from the following aspects:

1. An anti-EpCAM and CD3 bispecific antibody comprising an antigen binding domain that specifically binds to EpCAM and an antigen binding domain that specifically binds to CD3,

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

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

(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, according to the Kabat sequence numbering system, the sequence of CDRL1 is shown as SEQ ID NO:32, the sequence of CDRL2 is shown as SEQ ID NO:33, the sequence of CDRL3 is shown as SEQ ID NO:34, the sequence of CDRH1 is shown as SEQ ID NO:35, the sequence of CDRH2 is shown as SEQ ID NO:36, and the sequence of CDRH3 is shown as SEQ ID NO:37; or

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

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

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

Preferably, according to the Kabat sequence numbering system and the CDR definition system, the sequence of CDRL1 is shown as SEQ ID NO:38, the sequence of CDRL2 is shown as SEQ ID NO:39, the sequence of CDRL3 is shown as SEQ ID NO:40, the sequence of CDRH1 is shown as SEQ ID NO:41, the sequence of CDRH2 is shown as SEQ ID NO:42, and the sequence of CDRH3 is shown as SEQ ID NO:43;

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

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

CDRH1, CDRH2 and CDRH3 contained in the heavy chain variable region shown in SEQ ID NO:50, and CDRL1, CDRL2 and CDRL3 contained in the light chain variable region shown in SEQ ID NO:51,

Preferably, according to the Kabat sequence numbering system, the sequence of CDRH1 is shown as SEQ ID NO:44, the sequence of CDRH2 is shown as SEQ ID NO:45, and the sequence of CDRH3 is shown as SEQ ID NO:46, the sequence of CDRL1 is shown as SEQ ID NO:47, the sequence of CDRL2 is shown as SEQ ID NO:48, and the sequence of CDRL3 is shown as SEQ ID NO:49; or

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

CDRH1, CDRH2 and CDRH3 contained in the heavy chain variable region shown in SEQ ID NO:58, and CDRL1, CDRL2 and CDRL3 contained in the light chain variable region shown in SEQ ID NO:59,

Preferably, according to the Kabat sequence numbering system, the sequence of CDRH1 is shown as SEQ ID NO:52, the sequence of CDRH2 is shown as SEQ ID NO:53, and the sequence of CDRH3 is shown as SEQ ID NO:54, the sequence of CDRL1 is shown as SEQ ID NO:55, the sequence of CDRL2 is shown as SEQ ID NO:56, and the sequence of CDRL3 is shown as SEQ ID NO:57; wherein the variants of the CDRs have 3, 2 or 1 amino acid differences with the corresponding CDRs, respectively, or have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity, respectively.

2. The bispecific antibody of item 1, 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):

(i) the heavy chain variable region shown in SEQ ID NO:14, the light chain variable region shown in SEQ ID NO:13; or

(ii) the heavy chain variable region shown in SEQ ID NO:16, the light chain variable region shown in SEQ ID NO:15; and

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

(1) the heavy chain variable region shown in SEQ ID NO:50 and the light chain variable region shown in SEQ ID NO:51, or

(2) the heavy chain variable region shown in SEQ ID NO:58 and the light chain variable region shown in SEQ ID NO:59;

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 a ScFv.

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):

(i) the heavy chain variable region shown in SEQ ID NO:14, the light chain variable region shown in SEQ ID NO:13; and

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

(1) ScFv shown in SEQ ID NO: 18 or a variant thereof,

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

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.

3. The bispecific antibody of item 1 or 2, wherein the bispecific antibody comprises

(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 of the same; 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 NO: 1 and SEQ ID NO: 60-65), 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 NO: 6, SEQ ID NO: 7, and SEQ ID NO: 66-71) and CH3a;

(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 the N-terminus to the 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 the N-terminus to the 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 NO:6, SEQ ID NO:7, SEQ ID NO:66-71) 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);

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;

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 in Table 3;

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, which are replaced by an amino acid residue that has a positive charge under physiological conditions, and the other CH3 domain comprises one or more substitutions, which are replaced by 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 of may be aspartic acid (D) or glutamic acid (E), for example, the replaced amino acid residues include one or more of D356, L368, K392, D399 and K409, for example, one or more replacements in Table 4, 2) the replacement forms a disulfide bond between the heavy chain and the fusion peptide, for example, the replacement in Table 5, and/or 3) the replacement results in a significant decrease in the binding ability between Fc and protein A, for example, H435 and Y436 on a CH3 domain are replaced with arginine and phenylalanine, respectively, as shown in Table 6;

Preferably, wherein:

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

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

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

d) CH3b of the fusion peptide and CH3a of the heavy chain have substitutions that result in decreased ability to bind to protein A;

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 and SEQ ID NOs: 60-65;

Preferably, the first Fc fragment and/or the second Fc fragment comprises a CH2 of a sequence selected from any one of SEQ ID Nos: 6, SEQ ID NO: 7, SEQ ID NO: 66-71 and/or a CH3 of a sequence selected from any one of SEQ ID Nos: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 72-76;

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

(1) One of the sequences is shown as SEQ ID NO:8, and the other sequence is shown as SEQ ID NO:11;

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

(3) one of the sequences is shown as SEQ ID NO:72, and the other sequence is shown as SEQ ID NO:74;

(4) one of the sequences is shown as SEQ ID NO:9, and the other sequence is shown as SEQ ID NO:75;

(5) one of the sequences is shown as SEQ ID NO:73, and the other sequence is shown as SEQ ID NO:76;

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

(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; the light chain comprises or consists of SEQ ID NO:13 and SEQ ID NO:1;

(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; the light chain comprises or consists of SEQ ID NO:13 and SEQ ID NO:1;

(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; the light chain comprises or consists of SEQ ID NO:15 and SEQ ID NO:1;

(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; the light chain comprises or consists of SEQ ID NO:15 and SEQ ID NO:1;

(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;

(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;

(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;

(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;

(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; the light chain comprises or consists of SEQ ID NO:15 and SEQ ID NO:1;

(10) 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: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;

(11) 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: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; the light chain comprises or consists of SEQ ID NO:13 and SEQ ID NO:1; and

(12) It comprises a fusion peptide, a heavy chain and a light chain, or is composed 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 is composed 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 is composed of them; the light chain comprises SEQ ID NO:13 and SEQ ID NO:1, or is composed of them.

4. The bispecific antibody of any one of items 1-3, which is contained in a conjugate or fusion protein, wherein the bispecific antibody is conjugated or fused to a substance A, and the substance A is selected from a therapeutic agent, a drug precursor, a protein (such as an enzyme), a virus, a lipid, a biological response modifier (such as an immunomodulator), PEG, a hormone, an oligonucleotide, a diagnostic agent, a cytotoxic agent (which may be a drug or a toxin), an ultrasound enhancer, a non-radioactive marker, a detectable marker, such as a chemiluminescent labeling compound (such as luminol, isoluminol, thermal acridinium esters, imidazoles, acridinium salts and oxalate esters), or a fluorescent luminescent metal (such as 152Eu, or a lanthanide label).

In some embodiments, the ratio of the anti-EpCAM and CD3 bispecific antibody to the immune cells is 0.05 μg-27 μg anti-EpCAM and CD3 bispecific antibody/1×10 ⁶ immune cells, preferably 0.05 μg anti-EpCAM and CD3 bispecific antibody/1×10 ⁶ immune cells, 0.5 μg anti-EpCAM and CD3 bispecific antibody/1×10 ⁶ immune cells, 1 μg anti-EpCAM and CD3 bispecific antibody/1×10 ⁶ immune cells, 3 μg anti-EpCAM and CD3 bispecific antibody/1×10 ⁶ immune cells, 9 μg anti-EpCAM and CD3 bispecific antibody/1×10 ⁶ immune cells or 27 μg anti-EpCAM and CD3 bispecific antibody/1×10 ⁶ immune cells.

In some embodiments, the tumor is an EpCAM-positive tumor, such as colorectal cancer, gastric cancer, breast cancer, ovarian cancer, lung cancer (such as non-small cell lung cancer), prostate cancer, pancreatic cancer, liver cancer, retinoblastoma, esophageal cancer, kidney cancer, renal clear cell tumor, skin squamous cell carcinoma, skin basal cell carcinoma, sarcoma, nasal glioma, craniopharyngioma, thyroid cancer, cholangiocytoma, bladder cancer, head and neck tumors, cervical cancer, or oral cancer, etc.

The terms involved in the present invention have the conventional meanings understood by those skilled in the art. When a term has two or more definitions as used and/or accepted in the art, the definition of the term used herein is intended to include all meanings.

Those of ordinary skill in the art will appreciate that the CDR region of an antibody is responsible for the binding specificity of the antibody to an antigen. In the case of known antibody heavy chain and light chain variable region sequences, there are currently several methods for determining the CDR region of an antibody, including Kabat, IMGT, Chothia and AbM numbering systems. However, the application of each definition of the CDR of an antibody or its variants will be within the scope of the terms defined and used herein. If the variable region amino acid sequence of the antibody is given, then those skilled in the art can usually determine a specific CDR, without relying on any experimental data outside the sequence itself.

As used herein, "antibody" or "antigen binding fragment" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. The term "antibody" is used in a broad sense and includes immunoglobulin or antibody molecules, including monoclonal or polyclonal human, humanized, composite and chimeric antibodies and antibody fragments. Therefore, the term "antibody" includes any protein or peptide containing a specific molecule that contains at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen. Examples of this include, but are not limited to, the complementary determining region (CDR) of a heavy chain or light chain or its ligand binding portion, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region or any portion thereof, or at least a portion of a binding protein. In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human parts, can be prepared using DNA recombinant techniques well known in the art. The immunoglobulin molecules or antibody molecules of the present application may be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecules.

The term "antibody fragment" or "antigen-binding fragment" includes, but is not limited to, F(ab')2, F(ab)2, Fab', Fab, Fv, Fd, dAb, Fab/c, complementarity determining region (CDR) fragments, single-chain Fvs (ScFv), disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), diabody, disulfide-stabilized diabody (ds-Diabody), ScFv multimers (such as ScFv dimer, ScFv trimer), multispecific antibodies formed from a portion of an antibody comprising one or more CDRs, nanobodies, single domain antibodies (sdab), domain antibodies, bivalent domain antibodies, or any other antibody fragment that binds to an antigen but does not contain a complete antibody structure. Regardless of the structure, an antigen-binding fragment includes any polypeptide or polypeptide complex that is capable of binding to the same antigen as the parent antibody or parent antibody fragment. The term "antibody fragment" includes aptamers, spiegelmers and diabodies. The term "antibody fragment" also includes any synthetic or genetically modified protein that, like an antibody, can bind to a specific antigen to form a complex. Typically, an antibody fragment has at least about 50 consecutive amino acids of an antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

"Single chain variable fragment" or "ScFv" refers to a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin. In certain aspects, these regions are connected with a short linker peptide of 10 to about 25 amino acids. The linker can be rich in glycine for flexibility, and also contain serine or threonine for solubility, and can connect the N-terminus of VH to the C-terminus of VL, and vice versa. The protein retains the properties of the original immunoglobulin, except that the constant region has been removed and a linker has been introduced. ScFv molecules are known in the art, such as those described in U.S. Pat. No. 5,892,019.

The antigen binding domain that binds to EpCAM and CD3 is Fab, or ScFv, or non-covalent pairing (Fv) between the heavy chain variable region (VH) and the light chain variable region (VL). Any of the above antibodies or polypeptides may also include additional polypeptides, for example, a signal peptide at the N-terminus of the antibody, which is used to guide secretion, or other heterologous polypeptides as described herein, such as a 6×His tag for purification. The present invention includes not only complete antibodies, but also antibody fragments with immunological activity or fusion proteins formed by antibodies and other sequences. The present invention also provides other proteins or fusion expression products having the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having heavy and light chains containing variable regions, as long as the variable region is identical to the variable region of the heavy chain and light chain of the antibody of the present invention or at least 90% homology, preferably at least 95% homology, and optimally 96%, 97%, 98% or 99% homology or more. Therefore, the present invention includes those molecules having monoclonal antibody light chain and heavy chain variable regions with CDRs, as long as their CDRs have more than 90% (preferably more than 95%, most preferably more than 96%, 97%, 98% or 99%) homology with the CDRs of the present invention.

The present invention also includes fragments, variants, derivatives and analogs of the antibody. Antibodies, Fabs, variants or derivatives of the present application, include but are not limited to, polyclonal antibodies, monoclonal antibodies, multispecific antibodies (such as bispecific antibodies, trispecific antibodies, etc.), human antibodies, animal-derived antibodies, humanized antibodies, primatized (primatized) antibodies or chimeric antibodies, CDR grafted and/or modified antibodies, single-chain antibodies (e.g., ScFv), double-chain antibodies, antigen epitope binding fragments, e.g., Fab, Fab' and F(ab')2, Fd, Fv, single-chain Fv (ScFv), single-chain antibodies, disulfide-linked Fv (dsFv), fragments comprising VL domains or VH domains, fragments produced by Fab expression libraries, and anti-idiotypic (anti-Id) antibodies. The antibody fragment, antigen-binding fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substitution group in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a 6×His tag). According to the teachings herein, these fragments, derivatives and analogs are within the scope known to those skilled in the art.

The antibody of the present invention refers to a polypeptide having human EpCAM and CD3 binding activity and including the above-mentioned CDR region. The term also includes variant forms of polypeptides having the same function as the antibody of the present invention and including the above-mentioned CDR region. These variant forms include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions, insertions and/or substitutions, and addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, when substitution is made with amino acids with similar or similar properties, the function of the protein is generally not changed. For another example, adding one or several amino acids at the C-terminus and/or N-terminus generally does not change the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the present invention. Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

The antibody of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) a polypeptide having a substitution group in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a 6His tag). According to the teachings herein, these fragments, derivatives and analogs are within the scope known to those skilled in the art.

"Conservative amino acid substitutions" are those in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, non-essential amino acid residues of immunoglobulin polypeptides are preferably replaced by other amino acid residues from the same side chain family. In other embodiments, a string of amino acids may be replaced by a structurally similar string of amino acids that differ in sequence and/or in the composition of the side chain family.

Non-limiting examples of conservative amino acid substitutions are provided in Table 1 below, where a similarity score of 0 or higher indicates a conservative substitution between the two amino acids.

Table 1.

The C G P S A T D E N Q H K R V M I L F Y W W -8 -7 -6 -2 -6 -5 -7 -7 -4 -5 -3 -3 2 -6 -4 -5 -2 0 0 17 Y 0 -5 -5 -3 -3 -3 -4 -4 -2 -4 0 -4 -5 -2 -2 -1 -1 7 10 The F -4 -5 -5 -3 -4 -3 -6 -5 -4 -5 -2 -5 -4 -1 0 1 2 9 The The L -6 -4 -3 -3 -2 -2 -4 -3 -3 -2 -2 -3 -3 2 4 2 6 The The The I -2 -3 -2 -1 -1 0 -2 -2 -2 -2 -2 -2 -2 4 2 5 The The The The M -5 -3 -2 -2 -1 -1 -3 -2 0 -1 -2 0 0 2 6 The The The The The V -2 -1 -1 -1 0 0 -2 -2 -2 -2 -2 -2 -2 4 The The The The The The R -4 -3 0 0 -2 -1 -1 -1 0 1 2 3 6 The The The The The The The K -5 -2 -1 0 -1 0 0 0 1 1 0 5 The The The The The The The The H -3 -2 0 -1 -1 -1 1 1 2 3 6 The The The The The The The The The Q -5 -1 0 -1 0 -1 2 2 1 4 The The The The The The The The The The N -4 0 -1 1 0 0 2 1 2 The The The The The The The The The The The E -5 0 -1 0 0 0 3 4 The The The The The The The The The The The The D -5 1 -1 0 0 0 4 The The The The The The The The The The The The The T -2 0 0 1 1 3 The The The The The The The The The The The The The The A -2 1 1 1 2 The The The The The The The The The The The The The The The S 0 1 1 1 The The The The The The The The The The The The The The The The P -3 -1 6 The The The The The The The The The The The The The The The The The G -3 5 The The The The The The The The The The The The The The The The The The C 12 The The The The The The The The The The The The The The The The The The The

In some embodiments, the conservative substitution is preferably a substitution in which an amino acid within the following groups (a)-(e) is replaced by another amino acid residue within the same group: (a) small aliphatic, non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, non-polar residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

Particularly preferred conservative substitutions are as follows: Ala is replaced by Gly or by Ser; Arg is replaced by Lys; Asn is replaced by Gln or by His; Asp is replaced by Glu; Cys is replaced by Ser; Gln is replaced by Asn; Glu is replaced by Asp; Gly is replaced by Ala or by Pro; His is replaced by Asn or by Gln; Ile is replaced by Leu or by Val; Leu is replaced by Ile or by Val; Lys is replaced by Arg, replaced by Gln or by Glu; Met is replaced by Leu, replaced by Tyr or by Ile; Phe is replaced by Met, replaced by Leu or by Tyr; Ser is replaced by Thr; Thr is replaced by Ser; Trp is replaced by Tyr; Tyr is replaced by Trp; and/or Phe is replaced by Val, replaced by Ile or by Leu.

Fc amino acid numbering follows the Kabat numbering. "Kabat numbering" refers to the numbering system described by Kabat et al., which is recorded in the U.S. Department of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). The specific numbering is shown in Table 2 below:

Table 2. Fc amino acid numbering based on the Kabat numbering system

in,

Amino acids 221-227 are the hinge domain.

Amino acids 228-340 are the second constant region CH2 domain of the heavy chain.

Amino acids 341-447 are the third constant region CH3 domain of the heavy chain.

The antibodies can be modified to improve heterodimer pairing efficiency. For example, in some aspects, the heavy chain Fc fragment of the monovalent unit and/or the Fc fragment of the fusion peptide can contain one or more substitutions compared to the wild-type antibody fragment, which form a knob-hole structure pair. Knob-hole configurations are known in the art. See, for example, Ridgway et al., "Knob-into-holes" engineering of antibody CH3 domains for heavy chain heterodimerization, Protein Engineering 9(7):617-21 (1996).

In one aspect, T366 on one CH3 domain is replaced by a relatively large amino acid residue, such as tyrosine (Y) or tryptophan (W). Then, Y407 on the other CH3 domain can be replaced by a relatively small amino acid residue, such as threonine (T), alanine (A) or valine (V).

Table 3. Combinations of Fc amino acid substitutions form a knob-hole structure between the monovalent unit and the single-chain unit to improve heterodimer pairing efficiency

Combination number A replacement on CH3 Replacement on another CH3 1 T366W Y407A 2 T366W Y407V 3 T366Y Y407A 4 T366Y Y407V 5 T366W T366S, L368A, Y407V

In one aspect, one of the CH3 domains contains one or more substitutions with an amino acid residue that has a positive charge under physiological conditions, and the other CH3 domain contains one or more substitutions with one or more amino acid residues that have a negative charge under physiological conditions. In one aspect, the positively charged amino acid residue can be arginine (R), histidine (H) or lysine (K). In another aspect, the negatively charged amino acid residue can be aspartic acid (D) or glutamic acid (E). Amino acid residues that can be replaced include, but are not limited to, D356, L368, K392, D399 and K409.

Table 4. Combinations of CH3 amino acid substitutions form ionic bonds between monovalent units and single-chain units to improve heterodimer pairing efficiency

Combination number A replacement on CH3 Replacement on another CH3 1 D356K D399K K392D K409D 2 L368R D399K K392D K409D 3 L368K D399K K392D K409D 4 L368R D399K K409D 5 L368K D399K K409D 6 L368R K409D 7 L368K K409D

On one hand, S354 on one CH3 domain is replaced by cysteine, and Y349 on the other CH3 domain is also replaced by cysteine, and the residues at the two replaced positions form a disulfide bond.

Table 5. Combinations of CH3 amino acid substitutions to form disulfide bonds between monovalent units and single-chain units to improve heterodimer pairing efficiency

Combination number A replacement on CH3 Replacement on another CH3 1 S354C Y349C

On the one hand, H435 and Y436 on a CH3 domain were replaced with arginine and phenylalanine, respectively. This replacement resulted in a significant decrease in the binding ability between Fc and protein A, so that the heterodimer and homodimer had different protein A binding activities, making it easy to separate the two during affinity chromatography.

Table 6. One CH3 amino acid substitution results in decreased binding to protein A

Combination number Replacement on CH3 1 H435R, Y436F

In a preferred embodiment of the present invention, the CH3 amino acid sequence of the Fc forming the heterodimer is shown in Table 7 below:

Table 7.

In some embodiments, the antibodies of the present application include heterodimeric antibodies, which include two different antigen-binding polypeptide units. In some aspects, the heterodimer is different in size from its corresponding homodimer, and the difference in size can be used to facilitate the separation of heterodimers and homodimers.

In some aspects, as shown in Figure 1, one of the two antigen-binding polypeptide units comprises a light chain-heavy chain pair similar to a wild-type antibody. Throughout this application, this unit is also referred to as a "monovalent unit". In some aspects, as shown in Figure 1, the other antigen-binding polypeptide unit comprises a single-chain variable fragment (ScFv). Such ScFv can be fused to the N-terminus of the constant fragment (Fc) of the antibody, referred to as a fusion peptide. This fusion peptide is also referred to as a "single-chain unit" throughout this application.

Any of the above antibodies or polypeptides may also include additional polypeptides, for example, encoded polypeptides as described herein, signal peptides of antibody constant regions, the signal peptides being used to direct secretion, or other heterologous polypeptides as described herein. The antibodies described herein may be modified so that their amino acid sequences are different from the naturally occurring binding polypeptides from which they are derived. For example, the polypeptide or amino acid sequence from a specified protein may be similar to the starting sequence, for example, having a certain percentage of identity with the starting sequence, for example, it may have 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with the starting sequence. In addition, nucleotide or amino acid substitutions, deletions, or insertions may also be made to make conservative substitutions or changes in "non-essential" amino acid regions. For example, a polypeptide or amino acid sequence from a specified protein may be identical to the starting sequence, except for one or more independent amino acid substitutions, insertions, or deletions, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more independent amino acid substitutions, insertions, or deletions. In certain embodiments, a polypeptide or amino acid sequence from a specified protein has 1 to 5, 1 to 10, 1 to 15, or 1 to 20 independent amino acid substitutions, insertions, or deletions relative to the starting sequence.

The term "detectable label" as used herein refers to a compound or composition that can be detected directly or indirectly, and the compound or composition is directly or indirectly bound to a composition to be detected (e.g., a polynucleotide or protein, such as an antibody) to obtain a "labeled" composition. The term also includes a sequence that is bound to the polynucleotide, which provides a signal by the expression of the inserted sequence, such as green fluorescent protein (GFP) and the like. The label itself can be detected (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzyme label, can catalyze a chemical change in a substrate compound or composition that can be detected. The label can be used for small-scale detection or is more suitable for high-throughput screening. Similarly, suitable labels include, but are not limited to, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, and proteins (including enzymes). The label can be detected only or quantified. The reaction that is only detected generally includes a reaction that can only confirm its presence, wherein the reaction that can be quantified generally includes a reaction with a quantifiable (e.g., digitally reportable) value such as intensity, polarization and/or other properties. In luminescent or fluorescent assays, the detectable reaction can be directly using a luminophore or fluorophore associated with a component of the assay that actually involves binding, or indirectly using a luminophore or fluorophore linked to another (eg, a reporter molecule or indicator) component.

In some embodiments, the antibodies of the invention may be conjugated to therapeutic agents (e.g., chemotherapeutic agents, such as cisplatin, carboplatin), prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, or PEG. The antibodies of the invention may be linked to or fused to a therapeutic agent, which may include a detectable label, such as a radioactive label, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent, which may be a drug or toxin, an ultrasound enhancer, a non-radioactive label, combinations thereof, and other such components known in the art.

In certain embodiments, antigen-binding polypeptides include amino acid sequences or one or more groups that are not usually bound to antibodies. For example, the single-chain Fv antibody fragment of the present application may include a flexible linker sequence, or may be modified to add a functional group (e.g., polyethylene glycol (PEG), drugs, toxins, or markers). The antibody of the present application, its variant or derivative includes a modified derivative, that is, any type of molecule is covalently connected to the antibody and the covalent connection does not prevent the antibody from binding to the antigen epitope. In addition, the antibody may include one or more non-classical amino acids.

It should be noted that non-definite quantitative entity definitions should refer to one or more (kinds) of the entity; for example, "multifunctional antibody" should be understood to mean one or more (kinds) of multifunctional antibodies. Similarly, non-definite quantitative definitions, the terms "one or more" and "at least one" are used interchangeably herein.

The term "subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject, particularly a mammalian subject, for whom diagnosis, prognosis or treatment is desired. Mammalian subjects include humans, domestic animals, farm animals, zoos, playgrounds, or pets, such as dogs, cats, guinea pigs, rabbits, rats, mice, rats, horses, cattle, cows, primates (e.g., humans, monkeys such as cynomolgus monkeys, macaques, baboons, and chimpanzees, etc.), and the like.

As described herein, the antigen-binding polypeptides, variants or derivatives of the present application can be used in certain treatments and diagnostic methods related to cancer or infectious diseases. The application also relates to antibody-based treatments, which include administering the bispecific antibodies of the present application to patients, such as animals, mammals and humans, for the treatment of one or more diseases or conditions described herein. The therapeutic drugs of the present application include, but are not limited to, antibodies of the present application (including their variants and derivatives as described herein) and nucleic acids or polynucleotides encoding antibodies of the present application (including their variants and derivatives as described herein). The antibodies of the present application can also be used to treat, suppress or prevent diseases, disorders or conditions, including malignant diseases, disorders, or conditions associated with such diseases or disorders, such as diseases associated with immune responses. In some embodiments, the antibodies of the present invention can be used as immunosuppressants. In some embodiments, the antibodies of the present invention can be used to treat autoimmune diseases. The antigen-binding polypeptides of the present application, their variants or derivatives are used to suppress the growth, development and/or metastasis of cancer, particularly those listed above or listed in the following paragraphs.

The antibodies of the present application or their variants or derivatives can be used to treat, prevent, diagnose and/or predict other diseases or conditions associated with increased cell survival, including but not limited to cancer or tumors, including the development and/or metastasis of malignant tumors, and related diseases (such as malignant ascites, malignant pleural effusion, nausea and vomiting fluid), such as EpCAM-positive tumors.

The method of administering the antibody, its variant or derivative includes but is not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. The antibody or composition can be administered by any convenient route, such as by infusion or large dose injection, absorbed by the epithelium or mucosa and the inner layer of the skin (for example, oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other bioactive agents. Therefore, the pharmaceutical composition containing the antibody of the present application can be administered orally, rectally, parenterally, intracisternal, intravaginal, intraperitoneally, topically (such as through powders, ointments, drops or transdermal patches), buccally or as an oral or nasal spray. The term "parenteral" used herein refers to a mode of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion. Administration can be systemic or topical. It may also be desirable to administer the antigen-binding polypeptide or composition of the present application locally to the area in need of treatment, which may be achieved, for example but not limited to, by local perfusion during surgery, by topical application, for example in combination with a postoperative wound dressing, by injection, by catheter, by suppository, or by implant, wherein the implant is a porous, non-porous, or gel-like material, including a membrane or fiber. Preferably, when administering the protein (including antibodies) of the present application, care must be taken to use materials that do not absorb the protein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Schematic diagram of the YBODY antibody structure.

Figure 2. Detection of the binding effect between HCT116 and Jurkat cells mediated by bispecific antibodies. A: Negative control flow cytometric plot of HCT116+Jurkat without antibody, where Q1 is CFSE-stained Jurkat cells, Q2 is co-bound Jurkat and HCT116 cells, Q3 is PKH26-stained HCT116 cells, and Q4 is unstained cells; B: Flow cytometric plot of the HCT116+Jurkat+M701A 10μg/ml experimental group, the meaning of each quadrant is the same as before; C: Concentration gradient curves of HCT116 and Jurkat cell binding mediated by different antibodies.

Figure 3. Detection of biological activity of bispecific antibodies (reporter gene system).

Figure 4. In vitro killing assay mediated by bispecific antibodies. A: In vitro killing of B16-EpCAM by M701A; B: In vitro killing of B16 by M701A; C: In vitro killing of HCT116 by M701A; D: In vitro killing of OVCAR-3 by M701A; E: In vitro killing of CHO-K1-huEpCAM by different bispecific antibodies; F: In vitro killing of HCT116 by different bispecific antibodies.

Figure 5. In vivo efficacy of bispecific antibodies in the HCT116 human colon cancer model. A: Growth changes of mouse tumor volume; B: Changes in mouse body weight.

Figure 6. In vivo efficacy of bispecific antibodies in the OVCAR-3 human ovarian cancer model. A: Growth changes of mouse tumor volume; B: Changes in mouse body weight.

Figure 7. Pictures of purified TIL cells.

Figure 8. Proliferation curve of non-small cell lung cancer TIL cells.

Figure 9. Proliferation curve of breast cancer TIL cells.

Figure 10. Proliferation curve of cervical cancer TIL cells.

Figure 11. Melanoma TIL cell proliferation curve.

Figure 12. Cytotoxic activity of TIL+M701A cells and TIL cells against HCC827 tumor cells (detected by MTS method).

Figure 13. Cytotoxic activity of TIL+M701A cells and TIL cells against HCC827 tumor cells (detected by LDHA method).

Figure 14. Cytotoxic activity of TIL+M701A cells and TIL cells against HCC827 tumor cells (detected by RTCA method).

Figure 15. In vivo antitumor activity of TIL+M701A cells. The blank group was injected with vehicle, and the TIL+M701A group was injected with TIL+M701A cells at a ratio of 1 μg/1×10 ⁶ cells (antibody/TIL cell ratio).

Figure 16. Cytotoxic activity of TIL+M701A cells and TIL cells against MDA-MB-453 tumor cells (detected by LDHA method).

Figure 17. Cytotoxic activity of TIL+M701A cells and TIL cells against MDA-MB-453 tumor cells (detected by MTS method).

Detailed ways

The method and application of the present invention are described below in conjunction with the accompanying drawings. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as belonging to the protection scope of the present invention.

Example 1: Construction of anti-EpCAM-anti-CD3 bispecific antibody and detection of EpCAM terminal cell affinity

1. Construction of anti-EpCAM-anti-CD3 bispecific antibody

The structure of the bispecific antibody targeting EpCAM and CD3 includes an anti-EpCAM binding region and an anti-CD3 binding region, the monovalent unit is a pair formed by the anti-EpCAM heavy chain and the light chain, and the single-chain unit is an anti-CD3 ScFv-Fc form, which is defined as a YBODY structure (as shown in Figure 1), wherein the anti-CD3 VL is connected to the hinge region and CH2 through a linker. The heavy chain Fc of the monovalent unit and the Fc of the single-chain unit (with human IgG heavy chain Fc as the skeleton) are subjected to amino acid mutation modification, so that they are not easy to form homodimers (homodimer) but easy to form heterodimers (heterodimer). Using existing plasmids or synthetic gene fragments as templates, PCR and overlapping PCR are used to amplify the corresponding chains of the bispecific antibody, and each antibody chain is cloned into the pcDNA3.1 vector (Invitrogen) by enzyme cutting, connection or recombination. The specific sequence information of each antibody chain is shown in Table 8 and the sequence table.

Table 8. Amino acid sequence information corresponding to each antibody molecule

2. Bispecific Antibody EpCAM Cell Affinity Detection

The affinity of the antibody to human EpCAM on the cell surface was detected by FACS method using human colon cancer cell HCT116 (Shanghai Institutes for Biological Studies, Chinese Academy of Sciences) as the positive cell expressing human EpCAM on the cell membrane surface.

HCT116 cells were collected by centrifugation and resuspended in buffer (PBS + 1% FBS), and added to a 96-well plate at 2×10 ⁵ cells/well, 50 μL per well. After centrifugation at 350×g for 5 min, the supernatant was removed. The double antibody was diluted to 1000 nM with buffer, and then diluted in multiples, and then added to a 96-well plate at 50 μL/well. After resuspending, incubate in the dark for 1 hour, centrifuge and remove the supernatant, wash twice with buffer and resuspend in diluted PE-labeled anti-human IgG Fc antibody (Biolegend, 409304), incubate in the dark for 30 min, wash twice with buffer and resuspend in 100 μL buffer, and detect by flow cytometer (BD Accuri ^TM C6).

Each bispecific antibody had a significant binding effect with HCT116 cells, among which M700 was the anti-EpCAM monoclonal antibody control (light chain SEQ ID NO: 25 and heavy chain SEQ ID NO: 26). The specific EC50 values are shown in Table 9.

Table 9. Binding ability of bispecific antibodies to HCT116 cells

Antibody ID M700 M701 M701A M701B M701H M701I M701J M701K EC50(nM) 1.782 10.501 3.565 3.719 7.151 7.086 3.779 5.652

3. Bispecific antibody CD3 affinity detection (Biacore)

The human CD3 antigen (SB, Cat: CT038-H2508H) was fixed on the CM5 chip by amino coupling method, and the antigen coupling amount was 1500RU. When detecting the binding activity of the CD3 antigen end, the sample was diluted to the starting concentration with 1×HBS-EP+buffer, and then diluted to 4 concentrations by 2 times. The detection was performed from low concentration to high concentration on the machine, with a binding flow rate of 30μL/min, a binding time of 120s, and a dissociation time of 300s; the chip was regenerated with a pH1.5 Glycine solution, with a regeneration flow rate of 10μL/min and a regeneration time of 30s. After the detection, the result spectrum was fitted with the software Biacore T200 Evaluation Software in a 1:1Binding fitting mode to obtain the dissociation equilibrium constant (KD). As shown in Table 10, the series of bispecific antibodies all have binding effects with the human CD3 antigen. Among them, the anti-EpCAM antibody variable region sequences of M701A and M701B are the same (SEQ ID NO: 13 and SEQ ID NO: 14), and the anti-CD3 antibody variable region sequences are SEQ ID NO: 18 and SEQ ID NO: 19, respectively, with corresponding affinities of 21.15nM and 28.27nM, respectively. However, the variable region sequence of the anti-EpCAM antibody becomes another one (SEQ ID NO: 15 and SEQ ID NO: 16), and the variable region sequence of the anti-CD3 antibody is still SEQ ID NO: 18 and SEQ ID NO: 19, and the corresponding bispecific antibodies M701H and M701I have affinities of 95.26nM and 40.03nM, respectively. This shows that the affinity of the bispecific antibodies formed by the combination of different anti-EpCAM antibodies and different anti-CD3 antibodies is not regular.

Table 10. Biacore detection of the binding ability of each bispecific antibody to human CD3 antigen

Antibody ID M701 M701A M701B M701H M701I M701J M701K Kd(nM) 25.44 21.15 28.27 95.26 40.03 23.45 28.78

4. Bispecific antibody-mediated cell bridging

EpCAM-positive cell line HCT116 was stained with PKH26, and CD3-positive cell line Jurkat (Shanghai Institutes for Biological Studies, Chinese Academy of Sciences) was stained with CFSE. The stained cells were mixed and incubated with gradient dilutions of the test antibodies at a ratio of 1:1 (1×10 ⁵ HCT116 cells: 1×10 ⁵ Jurkat cells), where M700 was an anti-EpCAM monoclonal antibody control (light chain SEQ ID NO: 25 and heavy chain SEQ ID NO: 26), M100 was an anti-CD3 monoclonal antibody control (light chain SEQ ID NO: 27 and heavy chain SEQ ID NO: 28), and Mco101 was a bispecific antibody of anti-luciferase and anti-CD3, which served as the CD3-end isotype control of the bispecific antibody (light chain SEQ ID NO: 29, heavy chain SEQ ID NO: 30 and single chain SEQ ID NO: 31, with the same structure as in Figure 1). The cells were incubated for 1 hour, washed and resuspended, and then detected by flow cytometry (BD Accuri ^TM C6). The CFSE and PKH26 double-positive cells were the HCT116 and Jurkat cells bridged by the antibodies.

The results are shown in Figure 2. When Jurkat and HCT116 cells were mixed in a 1:1 ratio and hIgG, M700, M100, and Mco101 were added for 1 hour, HCT116 and Jurkat cells were not bridged; however, after the addition of M701A and M701B, the bridged cells accounted for about 40% of the total number of PKH26-positive cells, and the activity of M701A and M701B was equivalent. The cell interaction mediated by the bispecific antibody showed a positive dose-effect relationship with the antibody concentration.

5. Biological activity detection of bispecific antibodies (reporter gene method)

Jurkat-CD3-NFAT-RE-Luc cells (Promega) were used to detect the biological activity of the bispecific antibody. The pLV-puro (Inovogen Tech. Co., cat. No. VL3001) vector containing DNA encoding the human EpCAM gene (NCBI sequence number: NM_002354.3) was transfected into CHO-K1 cells to obtain the cell line CHO-K1-huEpCAM stably expressing human EpCAM. CHO-K1-huEpCAM was collected as target cells and resuspended in buffer (PBS + 1% FBS), added to a full white 96-well culture plate at 4×10 ⁴ cells/well, and placed in a 37°C, 5% CO2 incubator for overnight culture for 18 to 24 hours. The culture medium in the plate was removed, and 40 μL of antibody diluent was added to each well. Jurkat-CD3-NFAT-RE-Luc cells, i.e., effector cells, were taken out, the cells were blown off, and a single cell suspension was prepared. According to the effector-target ratio E:T=1.5:1, 40 μL was added to each well of an all-white 96-well culture plate, i.e., 6×10 ⁴ cells/well were plated. The all-white 96-well culture plate was placed in a 37°C, 5% CO2 incubator for 6 hours, 80 μL of Bio-Glo luciferase detection solution was added to each well, incubated at room temperature for 15 minutes away from light, placed in a multi-function reader, and the luminescence value was read by chemiluminescence.

The results are shown in Figure 3. In this reporter gene evaluation system, the bispecific antibodies M701, M701A, M701B, M701H, M701I, M701J, and M701K all showed biological activity, and the activities of M701A, M701J, and M701K were stronger than those of M701.

6. In vitro killing assay mediated by bispecific antibodies

The isolated PBMCs were used as effector cells and EpCAM-expressing cells were used as target cells to detect the in vitro killing effect mediated by the bispecific antibody. The pLV-puro (Inovogen Tech. Co., cat. No. VL3001) vector containing DNA encoding the human EpCAM gene (NCBI sequence number: NM_002354.3) was transfected into mouse melanoma cells B16 cells (Shanghai Institutes for Biological Studies, Chinese Academy of Sciences) to obtain a cell line B16-EpCAM that stably expresses human EpCAM, where B16 is used as a negative cell that does not express EpCAM. Other EpCAM-expressing target cells include human colon cancer cells HCT116 (Shanghai Institutes for Biological Studies, Chinese Academy of Sciences), human ovarian cancer cells OVCAR-3 (CCTCC, China Center for Type Culture Collection) and CHO-K1-huEpCAM. The cells were digested with trypsin to form a single cell suspension, centrifuged at 300g for 5min to collect the cells and stained with 5μM 5,6-carboxyfluorescein diacetate, succinimidyl ester (CFSE) (37°C, 15min), washed twice with complete medium, counted on a Vi-cell cell counter, and then added to a 96-well plate according to the experimental design, 2×10 ⁴ cells/100μL per well. The corresponding concentration of antibodies was added at 50μL/well, and hPBMCs were counted on a Cellometer cell counter and then added to a 96-well plate (2×10 ⁵ cells/50μL per well, with an effector-target ratio of 10:1). The cell culture plate was placed in a cell culture incubator and cultured for 72 h. After the cells were digested into a single cell suspension, a propidium bromide (PI) solution with a final concentration of 1 μg/mL was added. After incubation for 10 min, the cells were detected using a flow cytometer (BD Accuri ^TM C6) and the percentage of CFSE+PI+ double-positive cells to CFSE+ positive cells was analyzed.

As shown in Figures 4A and 4B, M701A only has a killing effect on B16-EpCAM cells expressing EpCAM, and has no killing effect on B16 cells without EpCAM expression, while the control antibody Mco101 has no killing effect on any cells, indicating that the bispecific antibody has a targeted effect. As shown in Figures 4C and 4D, M701A has a significant killing effect on both HCT116 and OVCAR-3 cells, and is stronger than the control Mco101. As shown in Figure 4E, the bispecific antibodies M701A, M701B, M701H, and M701I all have a significant killing effect on CHO-K1-huEpCAM cells, and the effects of M701A and M701B are significantly stronger than those of M701H and M701I. As shown in Figure 4F, both bispecific antibodies M701J and M701K had significant killing effects on HCT116 cells, with killing EC50 ranging from 7.814 to 25.43 ng/ml, and there was no significant difference between the killing level of M701J and M701A. The killing activity of the above bispecific antibodies was significantly stronger than that of M701 (EC50 of M701 was 69.17 ng/ml).

7. In vivo efficacy of bispecific antibodies in the HCT116 human colon cancer xenograft model

According to the culture conditions, sufficient amount of HCT116 cells and effector cells CIK (cytokine-induced killer, cytokine-induced killer cells, is a group of heterogeneous cells obtained by co-culturing human peripheral blood mononuclear cells with multiple cytokines in vitro for a period of time. Because this type of cell expresses two membrane protein molecules CD3+ and CD56+ at the same time, it is also called NK cell-like T lymphocytes), collect cells and count. Pre-mixed HCT116 cells (2×10 ⁶ cells/mouse) and CIK (2×10 ⁶ cells/mouse) were inoculated on the right back of each mouse, with an inoculation volume of 0.1 ml/mouse to establish a human colon cancer HCT116 xenograft tumor model. Drug treatment was started 1 hour after inoculation. The experiment was divided into the test drug M701A 2mg/kg group, M701A 1mg/kg group, M701 1mg/kg group, monoclonal antibody control M700 2mg/kg group and solvent control group (normal saline), with 8 mice in each group. The drug was administered by tail vein injection on days 0, 2, and 4 after inoculation, for a total of three times. The efficacy was evaluated based on the relative tumor inhibition rate and complete tumor regression rate, and the safety was evaluated based on the animal weight changes and mortality.

The formula for calculating tumor size is: tumor volume (mm ³ ) = 0.5 × (longest diameter of tumor × shortest diameter of tumor 2).

Relative tumor inhibition rate TGI (%): TGI = 1-T/C (%). T and C are the tumor volumes (TV) of the treatment group and the control group at a specific time point, respectively. The calculation formula is as follows: T/C% = TTV/CTV × 100% (TTV: average tumor volume of the treatment group; CTV: average tumor volume of the vehicle control group).

Complete regression rate of tumor: defined as the tumor volume being less than 63 mm ³ during or after treatment. Complete regression rate of tumor (%) = number of animals achieving complete regression in a group/total number of animals in the group×100%.

As shown in Figure 5A, the test drug M701A (2mg/kg, 1mg/kg) showed significant tumor inhibition effects on the 30th day after drug withdrawal in the treatment group (i.e., the 33rd day after inoculation), and the relative tumor inhibition rate TGI (%) was 100% and 93.82%, respectively. There were statistically significant differences relative to the vehicle control group (p values were all <0.001), and all tumors in the M701A (2mg/kg) group in both groups reached the standard of complete regression. The efficacy of M701A at these two doses was significantly better than that of M700 at 2mg/kg (p value <0.001), and the efficacy of M701A at the same dose was significantly better than that of M701 (both 1mg/kg). As shown in Figure 5B, no animal weight loss occurred during the treatment, and no drug toxicity was observed.

In the same tumor model, M701B, M701J and M701K showed similar tumor inhibition effects as M701A at the same dose, and there was no decrease in body weight.

8. In vivo efficacy of bispecific antibodies in the OVCAR-3 human ovarian cancer xenograft model

Sufficient amount of OVCAR-3 cells and effector cells CIK were cultured according to the culture conditions, and the cells were collected and counted. Pre-mixed OVCAR-3 cells (1×10 ⁷ cells/mouse) and CIK (1×10 ⁷ cells/mouse) were inoculated on the right back of each mouse, with an inoculation volume of 0.2 ml/mouse and a Matrigel gel content of 50% (0.1 ml/mouse) to establish a human ovarian cancer OVCAR-3 heterotopic transplant tumor model. Treatment was given 1h after inoculation. The experiment was divided into the test drug M701A (5 mg/kg), CD3 end isotype control Mco101 (5 mg/kg), monoclonal antibody control M700 (5 mg/kg) group and solvent control group (normal saline), with 8 mice in each group. The drug was injected into the tail vein, and the drug was administered on the 0th, 2nd and 4th days after inoculation, for a total of three times. The efficacy was evaluated according to the relative tumor inhibition rate (TGI) and the complete tumor regression rate, and the safety was evaluated according to the changes in animal weight and death.

As shown in Figure 6A, the test drug M701A (5 mg/kg) showed a significant tumor inhibition effect on the 44th day after drug withdrawal (i.e., the 48th day after inoculation) in the treatment group, and the relative tumor inhibition rate TGI (%) was 98.97%. There were statistically significant differences with respect to the solvent control group (p values were all <0.001), and the complete tumor regression rate of the M701A (5 mg/kg) group was 87.5%, and the efficacy of this group was significantly better than 5 mg/kg of M700 (TGI = 70.42%) (p value <0.001) and 5 mg/kg of Mco101 (TGI = 68.87%) (p value <0.001). As shown in Figure 6B, no animal weight loss occurred during the treatment, and no drug toxicity was observed.

In the same tumor model, M701B, M701J and M701K showed similar tumor inhibition effects as M701A at the same dose, and there was no decrease in body weight.

Example 2: Preparation of TIL cells

1. Purification of TIL cells

Place solid tumor tissue samples such as non-small cell lung cancer, breast cancer, cervical cancer, intestinal cancer, liver cancer, melanoma, gastric cancer, liver cancer, or ovarian cancer in a 100mm culture dish, cut them into 1-3mm tissue fragments in a digestion solution containing collagenase with medical scissors, and then transfer them to a centrifuge tube and place them in a 37℃ shaker for digestion for 1 hour. After digestion, remove them from the centrifuge tube, add an appropriate volume of X-VIVO15 culture medium (LONZA, 04-418Q), centrifuge and remove the supernatant, and finally resuspend the cell pellet with X-VIVO15 culture medium containing 1000-6000IU/ml IL-2, and inoculate them into a 24-well plate. Place the 24-well plate in a carbon dioxide incubator for static culture, replace fresh culture medium every 1-2 days, and if there are many cells, expand the wells. When the TIL cells cover the bottom of the plate, it indicates that the TIL cells have completely removed tumor cells, macrophages, fibroblasts, etc., and the TIL cells have been purified. See Figure 7 for purified TIL cells.

2. Expansion of TIL cells

The purified TIL cells can be activated using CD3 antibodies and irradiated PBMC cells. The specific operation is as follows: adjust the TIL cell density to 1-2×10 ⁶ cells/ml with X-VIVO15 medium, add 1000-6000IU/ml IL-2 (Sihuan Biological, National Medicine Standard S10970015), 10-100ng/ml CD3 antibody (Takara, T210) and 10-20×10 ⁶ cells/ml irradiated (50-100Gray) PBMC cells, mix well and place in a carbon dioxide incubator for culture. Thereafter, add an appropriate volume of X-VIVO15 medium containing 1000-6000IU/ml IL-2 every 2-3 days. During the culture process, 1-5% AB serum (Gemini, 100-512) and immune serum substitute (Gibco, A25961) can be added according to the cell state. After the cells have been cultured for about 15 days, a sufficient number of TIL cells can be harvested. The proliferation curves of TIL cells obtained from various cancer tissues are shown in Figures 8-11.

Example 3. Killing effect of anti-EpCAM and CD3 double antibody-M701A antibody armed TIL cells

1. Preparation of M701A antibody-immune cell complex

Non-small cell lung cancer cell line HCC827 (Peking Union Medical College Cell Resource Center) and breast cancer MDA-MB-453 cells (Peking Union Medical College Cell Resource Center) were subcultured using appropriate culture media, and the cells were digested with trypsin and sampled and counted. A certain number of tumor cells were taken out and added with M701A antibody according to different working concentrations. After incubation at room temperature for 30 minutes, they were washed twice with PBS, and finally the cell pellet was resuspended with PBS, and Mouse anti-human IgG Fc (Biolegend, 366904) secondary antibody was added. After incubation at 4°C for 30 minutes, the cells were washed and resuspended, and the M701A antibody loading was detected on a flow cytometer (Beckman cytoflex S). The working concentration gradient of M701A antibody was as follows: 27μg/1×10 ⁶ cells, 9μg/1×10 ⁶ cells, 3μg/1×10 ⁶ cells, 1μg/1×10 ⁶ cells, 500ng/1×10 ⁶ cells, and 50ng/1×10 ⁶ cells.

After the non-small cell lung cancer or breast cancer tumor tissue was prepared into a single cell suspension, a single cell suspension of 1×10 ⁶ cells/ml was prepared with a culture medium containing 3000IU/ml IL-2 and inoculated into a 24-well plate. The cell culture state was observed every 2 to 3 days. After the TIL cells entered the logarithmic growth phase, a certain number of TIL cells were taken and incubated according to the above-mentioned M701A antibody working concentration. After incubation at room temperature for 30 minutes, the cells were washed twice with PBS, and finally the cell pellet was resuspended with PBS, and Mouse anti-human IgG Fc (Biolegend, 366904) secondary antibody was added. After incubation at 4°C for 30 minutes, the cells were washed and resuspended, and the M701A antibody loading was detected by flow cytometry (Beckman cytoflex S). The results are shown in Table 11.

Table 11. Antibody loading on cell surface at different working concentrations of M701A antibody

A. Cytotoxic effect of TIL cells armed with M701A antibody on HCC827 tumor cells

(1) MTS assay to detect in vitro killing effect

After the non-small cell lung cancer tumor tissue was prepared into a single cell suspension, a single cell suspension of 1×10 ⁶ cells/ml was prepared with a culture medium containing 3000IU/ml IL-2 and inoculated into a 24-well plate. The cell culture state was observed every 2 to 3 days. After the TIL cells entered the logarithmic growth phase, a certain number of TIL cells were taken and incubated at room temperature for 30 minutes according to the antibody working concentration of 1μg/1×10 ⁶ cells, then washed twice with PBS, and finally the cell pellet was resuspended with a culture medium containing 1000-3000IU/ml IL-2. At this time, the TIL cells were armed with M701A antibodies and became TIL+M701A cells.

Tumor cells HCC827 were seeded in 96-well plates at 8000 cells/well 24 hours in advance. The above-mentioned TIL+M701A antibody cells and TIL cells were seeded into 96-well plates with tumor cells at effector-target ratios of 20:1, 10:1, 5:1, and 1:1, and placed in a carbon dioxide incubator at 37°C to co-culture TIL+M701A cells and tumor cells for 24 hours. After the co-culture, the percentage of cytotoxic lymphocytes (CTLs) in TIL cells armed with M701A antibodies was calculated using the MTS method.

The results in Figure 12 show that the CTL of TIL cells armed with M701A antibody was higher than that of TIL group, whether under the condition of low efficiency target ratio of 1:1 or high efficiency target ratio of 20:1, but the highest CTL level was 44% under the condition of high efficiency target ratio of 20:1, while the CTL of TIL group was only 19%. The CTL values of TIL group and TIL+M701A group increased with the increase of target ratio, but TIL+M701A group showed a better gradient trend.

(2) LDHA method to detect killing effect in vitro

Take the TIL cells in the logarithmic growth phase in the above (1), incubate them at room temperature for 30 minutes at a working concentration of 1 μg/1×10 ⁶ cells of antibody, then wash them twice with PBS, and finally resuspend the cell pellet in culture medium containing 1000-3000 IU/ml IL-2. At this time, the TIL cells have been armed with M701A antibodies and become TIL+M701A cells.

Tumor cells HCC827 were seeded in 96-well plates at 8000 cells/well 24 hours in advance, and TIL+M701A cells and TIL cells were seeded into 96-well plates with tumor cells at effector-target ratios of 10:1, 5:1, and 1:1, respectively, and placed in a carbon dioxide incubator at 37°C to co-culture TIL+M701A cells and tumor cells for 24 hours. After co-culture, the percentage of cytotoxic lymphocytes (CTLs) in TIL+M701A cells was calculated using the LDHA method.

The results in Figure 13 show that the CTL values of TIL+M701A cells at 1:1, 5:1 and 10:1 effector-target ratios were 1.74 to 1.98 times that of the TIL group. This indicates that TIL+M701A cells have better anti-tumor activity against tumor cells. The CTL activity of both the TIL+M701A group and the TIL group showed a high dependence on the effector-target ratio.

(3) RTCA detection of in vitro killing effect

RTCA (Real Time Cellular Analysis) uses a special process to integrate a gold microelectrode sensor array at the bottom of each cell growth well in a cell culture plate to construct a cell impedance detection sensor system that can track changes in cell morphology, proliferation and differentiation in real time, dynamically and quantitatively. When cells adherent to the microelectrode surface cause changes in the impedance of the adherent electrode interface, this change is correlated with the real-time state change of the cell. The biological information related to the physiological function of the cell can be obtained through real-time detection of the impedance value.

Take the TIL cells in the logarithmic growth phase in the above (1), incubate them at room temperature for 30 minutes at a working concentration of 1 μg/1×10 ⁶ cells of antibody, then wash them twice with PBS, and finally resuspend the cell pellet in culture medium containing 1000-3000 IU/ml IL-2. At this time, the TIL cells have been armed with M701A antibodies and become TIL+M701A cells.

Tumor cells HCC827 were seeded in a 96-well plate at 10,000 cells/well 24 hours in advance. TIL+M701A antibody cells and TIL cells were seeded into the 96-well plate with tumor cells at effector-target ratios of 40:1, 20:1, 10:1, and 5:1, respectively. They were placed in a carbon dioxide incubator at 37°C for co-culture, and the survival of tumor cells was detected in real time.

The results in Figure 14 show that after TIL cells or TIL+M701A cells were added to the 96-well cells containing HCC827 tumor cells, within 2 hours of interaction, the killing activity of the TIL group was better than that of the TIL+M701A group. However, starting from 4 hours, the CTL value of the TIL+M701A group gradually increased, and when the effector-target ratio was 20:1, the CTL value of the TIL+M701A group reached 97.07%, while that of the TIL group was only 59.36%.

(4) In vivo antitumor activity

2×10 ⁶ HCC827 tumor cells were subcutaneously transplanted into NOG mice (Viton Liva), and TIL+M701A was taken and injected into the peritumoral area on D4.

Take the TIL cells in the logarithmic growth phase in (1) above, incubate at room temperature for 30 minutes according to the working concentration of 1μg/1×10 ⁶ cells antibody, then wash twice with injection saline, and finally resuspend the cell pellet with injection saline containing 1% human albumin, and adjust the cell density to 2.5×10 ⁷ cells/ml. At this time, the TIL cells have been armed with M701A antibodies to become TIL+M701A cells. Take 200μl (i.e. 5×10 ⁶ cells) and inject them into mice from the peritumoral site, and inject 4000IU IL-2 per mouse on the same day. The blank control group was injected with injection saline containing 1% human albumin.

The results in Figure 15 show that the tumor volume in the blank group mice has been on an increasing trend. Compared with the blank group, the tumor volume in the TIL+M701A group mice was significantly controlled, and there was no increase in tumor volume after administration of TIL+M701A.

B. The killing effect of TIL cells armed with M701A antibody on MDA-MB-453 tumor cells

After breast cancer tumor tissue was prepared into a single cell suspension, a single cell suspension of 1×10 ⁶ cells/ml was prepared with a culture medium containing 3000IU/ml IL-2 and inoculated into a 24-well plate. The cell culture state was observed every 2 to 3 days. After the TIL cells entered the logarithmic growth phase, a certain number of TIL cells were taken and incubated at room temperature for 30 minutes at a working concentration of 1μg/1×10 ⁶ cells antibody, then washed twice with PBS, and finally resuspended with a culture medium containing 1000-3000IU/ml IL-2. At this time, the TIL cells were armed with M701A antibodies and became TIL+M701A cells.

Tumor cells MDA-MB-453 were seeded in 96-well plates at 8000 cells/well 24 hours in advance, and the above TIL cells were seeded into 96-well plates with tumor cells at effector-target ratios of 40:1, 20:1, 10:1, 5:1, 2.5:1, and 1:1, respectively, and placed in a carbon dioxide incubator at 37°C to co-culture TIL+M701A group cells and TIL group cells and tumor cells for 24 hours. After the co-culture, the percentage of cytotoxic lymphocytes (CTLs) in TIL+M701A cells was calculated using the MTS method and LDHA method.

The results are shown in Figure 16 (MTS method) and Figure 17 (LDHA method): Both methods of detecting the killing activity of TIL cells against tumor cells showed that the anti-tumor activity increased with the increase of the effector-target ratio. At the same effector-target ratio, the anti-tumor activity of the TIL+M701A group was 1 to 2 times that of the TIL group. This phenomenon was more significant at a low effector-target ratio.

In summary, after TIL cells are armed with the M701 series antibodies, the spatial distance between tumor cells and T cells can be shortened, and TIL cells can be activated, which can effectively enhance the anti-tumor activity of TIL cells.

Sequence Listing

Claims (13)

A bispecific antibody and an immune cell complex, wherein the bispecific antibody is an anti-EpCAM and CD3 bispecific antibody and comprises an antigen binding domain that specifically binds to EpCAM and an antigen binding domain that specifically binds to CD3, and the immune cell binds to the anti-EpCAM and CD3 bispecific antibody, Preferably, the immune cells are selected from TIL cells, CIK cells, LAK cells, T cells, and NKT cells. The bispecific antibody and immune cell complex according to claim 1, wherein the immune cell is a TIL cell. The bispecific antibody and immune cell complex according to any one of claims 1 to 2, The antigen binding domain that specifically binds to EpCAM is selected from the group consisting of: 1) An antigen-binding domain that specifically binds to EpCAM and comprises the following CDRs or variants thereof: (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, according to the Kabat sequence numbering system, the sequence of CDRL1 is shown as SEQ ID NO:32, the sequence of CDRL2 is shown as SEQ ID NO:33, the sequence of CDRL3 is shown as SEQ ID NO:34, the sequence of CDRH1 is shown as SEQ ID NO:35, the sequence of CDRH2 is shown as SEQ ID NO:36, and the sequence of CDRH3 is shown as SEQ ID NO:37; or 2) an antigen-binding domain that specifically binds to EpCAM and comprises the following CDRs or variants thereof: (i) CDRH1, CDRH2 and CDRH3 contained in the heavy chain variable region shown in SEQ ID NO:16, and (ii) CDRL1, CDRL2 and CDRL3 contained in the light chain variable region shown in SEQ ID NO:15, Preferably, according to the Kabat sequence numbering system and the CDR definition system, the sequence of CDRL1 is shown as SEQ ID NO:38, the sequence of CDRL2 is shown as SEQ ID NO:39, the sequence of CDRL3 is shown as SEQ ID NO:40, the sequence of CDRH1 is shown as SEQ ID NO:41, the sequence of CDRH2 is shown as SEQ ID NO:42, and the sequence of CDRH3 is shown as SEQ ID NO:43; and The antigen binding domain that specifically binds to CD3 is selected from the group consisting of: 1) An antigen-binding domain that specifically binds to CD3 comprising the following CDRs or variants thereof: CDRH1, CDRH2 and CDRH3 contained in the heavy chain variable region shown in SEQ ID NO:50, and CDRL1, CDRL2 and CDRL3 contained in the light chain variable region shown in SEQ ID NO:51, Preferably, according to the Kabat sequence numbering system, the sequence of CDRH1 is shown as SEQ ID NO:44, the sequence of CDRH2 is shown as SEQ ID NO:45, and the sequence of CDRH3 is shown as SEQ ID NO:46, the sequence of CDRL1 is shown as SEQ ID NO:47, the sequence of CDRL2 is shown as SEQ ID NO:48, and the sequence of CDRL3 is shown as SEQ ID NO:49; or 2) an antigen-binding domain that specifically binds to CD3 comprising the following CDRs or variants thereof: CDRH1, CDRH2 and CDRH3 contained in the heavy chain variable region shown in SEQ ID NO:58, and CDRL1, CDRL2 and CDRL3 contained in the light chain variable region shown in SEQ ID NO:59, Preferably, according to the Kabat sequence numbering system, the sequence of CDRH1 is shown as SEQ ID NO:52, the sequence of CDRH2 is shown as SEQ ID NO:53, and the sequence of CDRH3 is shown as SEQ ID NO:54, the sequence of CDRL1 is shown as SEQ ID NO:55, the sequence of CDRL2 is shown as SEQ ID NO:56, and the sequence of CDRL3 is shown as SEQ ID NO:57; wherein the variants of the CDRs have 3, 2 or 1 amino acid differences with the corresponding CDRs, respectively, or have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity, respectively. The bispecific antibody and immune cell complex according to any one of claims 1 to 3, 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): (i) the heavy chain variable region shown in SEQ ID NO:14, the light chain variable region shown in SEQ ID NO:13; or (ii) a heavy chain variable region as shown in SEQ ID NO: 16, and a light chain variable region as shown in SEQ ID NO: 15; and wherein the antigen binding domain that specifically binds to CD3 comprises the following heavy chain variable region and light chain variable region (or variants thereof): (1) the heavy chain variable region shown in SEQ ID NO:50 and the light chain variable region shown in SEQ ID NO:51, or (2) the heavy chain variable region shown in SEQ ID NO:58 and the light chain variable region shown in SEQ ID NO:59; 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 a ScFv. 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): (i) the heavy chain variable region shown in SEQ ID NO:14, the light chain variable region shown in SEQ ID NO:13; and wherein the antigen binding domain that specifically binds to CD3 is selected from the group consisting of: (1) ScFv shown in SEQ ID NO: 18 or a variant thereof, (2) ScFv shown in SEQ ID NO: 19 or a variant thereof, 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. The bispecific antibody and immune cell complex according to any one of claims 1 to 4, wherein the bispecific antibody comprises (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 of the same; 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 NO: 1 and SEQ ID NO: 60-65), 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 NO: 6, SEQ ID NO: 7, and SEQ ID NO: 66-71) and CH3a; and (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 the N-terminus to the 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 the N-terminus to the 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 NO:6, SEQ ID NO:7, SEQ ID NO:66-71) 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); 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; 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 in Table 3; 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, which are replaced by an amino acid residue that has a positive charge under physiological conditions, and the other CH3 domain comprises one or more substitutions, which are replaced by 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 of may be aspartic acid (D) or glutamic acid (E), for example, the replaced amino acid residues include one or more of D356, L368, K392, D399 and K409, for example, one or more replacements in Table 4, 2) the replacement forms a disulfide bond between the heavy chain and the fusion peptide, for example, the replacement in Table 5, and/or 3) the replacement results in a significant decrease in the binding ability between Fc and protein A, for example, H435 and Y436 on a CH3 domain are replaced with arginine and phenylalanine, respectively, as shown in Table 6; Preferably, wherein: a) CH3b of the fusion peptide and CH3a of the heavy chain have a substitution pair that forms a knob-hole structure; b) CH3b of the fusion peptide and CH3a of the heavy chain have a substitution pair that forms an ionic bond; c) CH3b of the fusion peptide and CH3a of the heavy chain have a substitution pair that forms a disulfide bond; and/or d) CH3b of the fusion peptide and CH3a of the heavy chain have substitutions that result in decreased ability to bind to protein A; 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 and SEQ ID NOs: 60-65; Preferably, the first Fc fragment and/or the second Fc fragment comprises a CH2 of a sequence selected from any one of SEQ ID Nos: 6, SEQ ID NO: 7, SEQ ID NO: 66-71 and/or a CH3 of a sequence selected from any one of SEQ ID Nos: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 72-76; Preferably, the sequences of CH3a and CH3b are selected from the group consisting of: (1) One of the sequences is shown as SEQ ID NO:8, and the other sequence is shown as SEQ ID NO:11; (2) one of the sequences is shown as SEQ ID NO:9, and the other sequence is shown as SEQ ID NO:12; (3) one of the sequences is shown as SEQ ID NO:72, and the other sequence is shown as SEQ ID NO:74; (4) one of the sequences is shown as SEQ ID NO:9, and the other sequence is shown as SEQ ID NO:75; (5) One of the sequences is shown as SEQ ID NO:73, and the other sequence is shown as SEQ ID NO:76. The bispecific antibody and immune cell complex according to any one of claims 1 to 5, wherein the bispecific antibody is selected from the group consisting of: (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; the light chain comprises or consists of SEQ ID NO:13 and SEQ ID NO:1; (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; the light chain comprises or consists of SEQ ID NO:13 and SEQ ID NO:1; (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; the light chain comprises or consists of SEQ ID NO:15 and SEQ ID NO:1; (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; the light chain comprises or consists of SEQ ID NO:15 and SEQ ID NO:1; (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; the light chain comprises or consists of SEQ ID NO:13 and SEQ ID NO:1; (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; (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; the light chain comprises or consists of SEQ ID NO:13 and SEQ ID NO:1; (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; (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; (10) 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: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; (11) 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: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; the light chain comprises or consists of SEQ ID NO:13 and SEQ ID NO:1; and (12) It comprises a fusion peptide, a heavy chain and a light chain, or is composed 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 is composed 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 is composed of them; the light chain comprises SEQ ID NO:13 and SEQ ID NO:1, or is composed of them. The bispecific antibody and immune cell complex according to any one of claims 1 to 6, wherein the bispecific antibody is contained in a conjugate or a fusion protein, wherein the bispecific antibody is conjugated or fused to a substance A, and the substance A is selected from a therapeutic agent, a drug precursor, a protein (such as an enzyme), a virus, a lipid, a biological response modifier (such as an immunomodulator), PEG, a hormone, an oligonucleotide, a diagnostic agent, a cytotoxic agent (which may be a drug or a toxin), an ultrasound enhancer, a non-radioactive marker, a detectable marker, such as a chemiluminescent labeling compound (such as luminol, isoluminol, thermal acridinium esters, imidazoles, acridinium salts and oxalates), or a fluorescent luminescent metal (such as 152Eu, or a lanthanide label). The bispecific antibody and immune cell complex according to any one of claims 1 to 7, wherein the ratio of the anti-EpCAM and CD3 bispecific antibody to the immune cells is 0.05 μg-27 μg anti-EpCAM and CD3 bispecific antibody/1×10 ⁶ immune cells, preferably 0.05 μg anti-EpCAM and CD3 bispecific antibody/1×10 ⁶ immune cells, 0.5 μg anti-EpCAM and CD3 bispecific antibody/1×10 ⁶ immune cells, 1 μg anti-EpCAM and CD3 bispecific antibody/1×10 ⁶ immune cells, 3 μg anti-EpCAM and CD3 bispecific antibody/1×10 ⁶ immune cells, 9 μg anti-EpCAM and CD3 bispecific antibody/1×10 ⁶ immune cells or 27 μg anti-EpCAM and CD3 bispecific antibody/1×10 ⁶ immune cells. A pharmaceutical composition comprising the bispecific antibody and immune cell complex according to any one of claims 1 to 8, and a pharmaceutically acceptable carrier. A kit comprising an immune cell and an anti-EpCAM and CD3 bispecific antibody, or comprising a bispecific antibody and an immune cell complex according to any one of claims 1 to 8, preferably, the immune cell and the anti-EpCAM and CD3 bispecific antibody are as defined in any one of claims 1 to 8, preferably, the kit further comprises a drug (such as a small molecule drug or a macromolecule drug) for treating tumors and/or malignant ascites, malignant effusion, malignant pleural effusion, etc. A method for improving the killing activity of anti-EpCAM and CD3 bispecific antibodies and/or immune cells, the method comprising combining the immune cells with anti-EpCAM and CD3 bispecific antibodies, preferably culturing the immune cells with anti-EpCAM and CD3 bispecific antibodies, wherein the immune cells and the anti-EpCAM and CD3 bispecific antibodies are as defined in any one of claims 1 to 8. A method for treating tumors and/or malignant ascites, malignant effusion, malignant pleural effusion, etc., comprising administering to a subject a therapeutically effective amount of the bispecific antibody and immune cell complex according to any one of claims 1 to 8 or the pharmaceutical composition according to claim 9. The kit according to claim 10 or the method according to claim 12, wherein the tumor is an EpCAM-positive tumor, such as colorectal cancer, gastric cancer, breast cancer, ovarian cancer, lung cancer (such as non-small cell lung cancer), prostate cancer, pancreatic cancer, liver cancer, retinoblastoma, esophageal cancer, kidney cancer, renal clear cell tumor, skin squamous cell carcinoma, skin basal cell carcinoma, sarcoma, nasal glioma, craniopharyngioma, thyroid cancer, cholangiocytoma, bladder cancer, head and neck tumors, cervical cancer or oral cancer.

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