WO2021098822A1 - Bispecific antibodies - Google Patents

Abstract

The present disclosure relates to bispecific antibodies. Specifically, the present disclosure relates to anti-CEA/CD3 bispecific antibodies and an application thereof. The present disclosure relates to bispecific antibodies capable of specifically binding CEA and specifically binding CD3, a composition comprising the bispecific antibodies, and a use thereof as a medicament.

Description

A bispecific antibody

This application claims the priority of the Chinese patent application CN201911148796.4 whose filing date is November 21, 2019. This application quotes the full text of the aforementioned Chinese patent application.

Technical field

This disclosure relates to the field of antibody drugs. Specifically, it includes anti-CEA/CD3 bispecific antibody drugs and their applications.

Background technique

The statements here only provide background information related to this disclosure, and do not necessarily constitute prior art.

Carcinoembryonic antigen (CEA, also known as CEACAM-5 or CD66e) is one of the first tumor-associated antigens discovered by people. It is a glycoprotein with a molecular weight of about 180kDa. CEA is a member of the immunoglobulin superfamily and contains 7 domains connected to the cell membrane via glycosylphosphatidylinositol (GPI) anchors (Thompson J.A., J Clin Lab Anal. 5:344-366, 1991). CEA was first discovered and reported in colon cancer tissue extracts by Gold P and Freedman SO (Gold and Freedman 1965; Gold and Freedman 1965), and subsequently reported the use of sensitive radioimmunoassay methods in the serum of colon cancer patients and other tumor patients CEA was detected in the sera, and the CEA content in the serum of healthy people or patients with other diseases was extremely low (Thomson, Krupey et al. 1969). The expression of CEA is increased in cancer cells, and the increased CEA promotes cell adhesion, which in turn promotes cell metastasis (Marshall J., Semin Oncol., 30(增刊8): 30-6, 2003). CEA is commonly expressed in epithelial tissues, including cells in the gastrointestinal, respiratory and genitourinary tracts, and cells in the colon, cervix, sweat glands and prostate (Nap et al., Tumour Biol., 9(2-3): 145-53, 1988; Nap et al., Cancer Res., 52(8): 2329-23339, 1992). So far, see WO1999043817A1, WO2004032962A1, WO2005086875A3, WO2012117002A1 and other patent applications that disclose a variety of anti-CEA antibodies.

Bispecific T cell adaptor (BiTE) can simultaneously target tumor cell surface antigen and T cell surface CD3 antigen, using T immune cell redirection to achieve the effect of killing tumor cells. This type of bispecific antibody, marked by Blinatumomab, has undergone the first generation of BiTE without Fc, the second generation with Fc-containing symmetrical structure, and the current third-generation Fc-containing asymmetric structure has been modified and optimized. The half-life of the drug ensures the effectiveness while also improving the safety. The third-generation bispecific antibodies are represented by Roche’s TCB, including CD20-TCB and CEA-TCB. Through a 2:1 differential design, the affinity of tumor antigens is improved, while the affinity of CD3 is controlled. It is in preclinical in vivo Both the pharmacodynamic model and the latest clinical data show significant efficacy, and the side effects of CD3 have also been well controlled.

The shortcomings and deficiencies of the third-generation bispecific antibodies are mainly reflected in the large number of chains, the difficulty of controlling the expression ratio of different chains, and the complex molecular structure and serious mismatch problems, which pose a great challenge for downstream process development.

Summary of the invention

This disclosure provides a new bispecific antibody.

In some embodiments, the bispecific antibodies of the present disclosure comprise an anti-CEA antibody portion and an anti-CD3 antibody portion, wherein:

The anti-CEA antibody portion includes the heavy chain variable region and the light chain variable region of the anti-CEA antibody:

(a) The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID NOs: 7, 8 and 9, respectively, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region As shown in SEQ ID NO: 10, 11 and 12 respectively; or

(b) The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID NOs: 13, 14 and 15, respectively, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region As shown in SEQ ID NO: 16, 17 and 18 respectively; or

(c) The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID NOs: 7, 19 and 9, respectively, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region As shown in SEQ ID NO: 10, 11, and 12, respectively.

In some embodiments, the anti-CD3 antibody portion of the bispecific antibody of the present disclosure includes the heavy chain variable region and the light chain variable region of an anti-CD3 antibody, wherein:

The amino acid sequences of HCDR1, HCDR2, and HCDR3 of the heavy chain variable region are as shown in SEQ ID NOs: 37, 38, and 39, respectively, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 of the light chain variable region are as shown in SEQ ID NO: 40, 41 and 42 are shown.

In some embodiments, the anti-CEA antibody portion of the bispecific antibody of the present disclosure comprises a heavy chain variable region and a light chain variable region as shown below, wherein:

(d) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 20, or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 20; and/or

The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 21, or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 21; or

(e) The amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO: 22, or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 22; and/or

The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 23, or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 23.

In some embodiments, the aforementioned (d) or (e) having at least 90% sequence identity refers to having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% or 100% sequence identity.

In some embodiments, the anti-CEA antibody portion of the bispecific antibody of the present disclosure comprises a heavy chain variable region and a light chain variable region as shown below, wherein:

The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 20; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 21; or

The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 22; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 23.

In some embodiments, the anti-CD3 antibody portion or the anti-CEA antibody portion of the bispecific antibody of the present disclosure is a scFv structure or a Fab fragment structure.

In some embodiments, the bispecific antibody as described above has an IgG-(scFv) 2 structure or an IgG-scFv structure.

In some embodiments, the bispecific antibody as described above, wherein the anti-CD3 antibody portion comprises the heavy chain variable region and the light chain variable region of an anti-CD3 antibody, wherein:

The heavy chain variable region of the anti-CD3 antibody has the same sequence as the heavy chain variable region contained in SEQ ID NO: 36, and

The light chain variable region of the anti-CD3 antibody has the same sequence as the light chain variable region contained in SEQ ID NO: 36.

In some embodiments, the anti-CD3 antibody portion of the bispecific antibody as described above comprises the heavy chain variable region and the light chain variable region of a CD3 antibody, wherein:

The heavy chain variable region of the anti-CD3 antibody is shown in SEQ ID NO: 52, and the light chain variable region of the anti-CD3 antibody is shown in SEQ ID NO: 51.

In some embodiments, the CEA antibody portion of the aforementioned bispecific antibody is an IgG type antibody, wherein the IgG type antibody comprises two heavy chains and two light chains.

In some embodiments, the CEA antibody portion of the aforementioned bispecific antibody is an IgG-type antibody, wherein the IgG-type antibody comprises two heavy chains with incomplete sequences and two light chains with identical sequences.

In some embodiments, the CEA antibody portion of the aforementioned bispecific antibody is an IgG-type antibody, wherein the IgG-type antibody comprises two heavy chains with identical sequences and two light chains with identical sequences.

In some embodiments, the anti-CEA antibody portion of the bispecific antibody as described above is an IgG antibody comprising an antibody light chain and an antibody heavy chain, wherein,

(f) The heavy chain includes a first heavy chain and a second heavy chain, and the amino acid sequences of the first heavy chain and the second heavy chain are shown in SEQ ID NOs: 28 and 29, or are different from SEQ ID NOs. : The amino acid sequences shown in 28 and 29 have at least 80% sequence identity; and

The light chain amino acid sequence is shown in SEQ ID NO: 31, or has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 31; or

(g) The amino acid sequence of the heavy chain is shown in SEQ ID NO: 30, or has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 30; and

The light chain amino acid sequence is shown in SEQ ID NO: 31, or has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 31; or

(h) The heavy chain includes a first heavy chain and a second heavy chain, and the amino acid sequences of the first heavy chain and the second heavy chain are shown in SEQ ID NOs: 32 and 33 respectively, or are different from SEQ ID NOs. : The amino acid sequences shown in 32 and 33 have at least 80% sequence identity; and

The light chain amino acid sequence is shown in SEQ ID NO: 35, or has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 35; or

(i) The heavy chain amino acid sequence is shown in SEQ ID NO: 34, or has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 34; and

The light chain amino acid sequence is shown in SEQ ID NO: 35, or has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 35.

In some embodiments, the aforementioned (f) to (i) having at least 80% sequence identity means having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

In some embodiments, the anti-CEA antibody portion of the bispecific antibody as described above is an IgG antibody, wherein:

(f-2) The anti-CEA antibody comprises two heavy chains with different sequences and two light chains with the same sequence, wherein the amino acid sequences of the first heavy chain and the second heavy chain are as shown in SEQ ID NO: 28 and 29, respectively. Or have at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 28 and 29, respectively; and the amino acid sequence of the light chain is shown in SEQ ID NO: 31, or with the amino acid sequence shown in SEQ ID NO: 31. The amino acid sequence shown has at least 80% sequence identity; or

(g-2) The anti-CEA antibody comprises two heavy chains with the same sequence and two light chains with the same sequence, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO: 30, or is the same as SEQ ID NO: 30 The amino acid sequence shown has at least 80% sequence identity; and the light chain amino acid sequence is shown in SEQ ID NO: 31, or has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 31; or

(h-2) The anti-CEA antibody comprises two heavy chains with different sequences and two light chains with the same sequence, wherein the amino acid sequences of the first heavy chain and the second heavy chain are as shown in SEQ ID NO: 32 and 33, respectively. Or have at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 32 and 33; and the light chain amino acid sequence is shown in SEQ ID NO: 35, or is shown in SEQ ID NO: 35 The amino acid sequence of has at least 80% sequence identity; or

(i-2) The anti-CEA antibody comprises two heavy chains with the same sequence and two light chains with the same sequence, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO: 34 or is the same as SEQ ID NO: 34 The amino acid sequence shown has at least 80% sequence identity; and the light chain amino acid sequence is shown in SEQ ID NO: 35, or has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 35.

In some embodiments, the anti-CEA antibody portion of the bispecific antibody as described above is an IgG antibody, and the IgG antibody comprises an antibody light chain and an antibody heavy chain, wherein,

(f-1) The heavy chain includes a first heavy chain and a second heavy chain, and the amino acid sequences of the first heavy chain and the second heavy chain are shown in SEQ ID NOs: 28 and 29, respectively; and The chain amino acid sequence is shown in SEQ ID NO: 31; or

(g-1) The heavy chain amino acid sequence is shown in SEQ ID NO: 30; and the light chain amino acid sequence is shown in SEQ ID NO: 31; or

(h-1) The heavy chain includes a first heavy chain and a second heavy chain, and the amino acid sequences of the first heavy chain and the second heavy chain are shown in SEQ ID NOs: 32 and 33, respectively; and The chain amino acid sequence is shown in SEQ ID NO: 35; or

(i) The heavy chain amino acid sequence is shown in SEQ ID NO: 34; and the light chain amino acid sequence is shown in SEQ ID NO: 35.

In some embodiments, the anti-CD3 antibody portion of the bispecific antibody as described above comprises the heavy chain variable region and the light chain variable region of a CD3 antibody, wherein:

The heavy chain variable region of the anti-CD3 antibody has the same sequence as the heavy chain variable region contained in SEQ ID NO: 36, and/or

The light chain variable region of the anti-CD3 antibody has the same sequence as the light chain variable region contained in SEQ ID NO: 36; preferably, the amino acid sequence of the heavy chain variable region of the anti-CD3 antibody is as SEQ ID NO : Shown in 52; and/or the amino acid sequence of the light chain variable region of the anti-CD3 antibody is shown in SEQ ID NO: 51.

In some embodiments, the bispecific antibody as described above, wherein the anti-CD3 antibody portion is an anti-CD3 single-chain antibody, and the amino acid sequence of the single-chain antibody is shown in SEQ ID NO: 36.

In some embodiments, in the bispecific antibody as described above, the heavy chain of the anti-CEA antibody and the anti-CD3 single-chain antibody are connected by a peptide bond or a linker.

In some embodiments, the bispecific antibody as described above, wherein the bispecific antibody has an IgG-(scFv)2 structure.

In some embodiments, the bispecific antibody chain 1 and chain 2 as described above, the chain 1 comprises VH(CEA)-CH1-Fc-scFv(CD3) from the amino terminus to the carboxy terminus, wherein VH(CEA )-CH1-Fc is fused to scFv (CD3) directly or via a linker; and

The chain 2 contains VL(CEA)-CL from the amino terminal to the carboxy terminal; preferably,

(j) The amino acid sequence of the chain 1 is shown in SEQ ID NO: 44, or has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 44, and

The amino acid sequence of the chain 2 is shown in SEQ ID NO: 31, or has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 31; or

(k) The amino acid sequence of the chain 1 is shown in SEQ ID NO: 46, or has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 46, and

The amino acid sequence of the chain 2 is shown in SEQ ID NO: 35, or has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 35.

In some embodiments, the bispecific antibody as previously described includes chain 1 and chain 2,

The amino acid sequence of the chain 1 is shown in SEQ ID NO: 44, and the amino acid sequence of the chain 2 is shown in SEQ ID NO: 31; or

The amino acid sequence of chain 1 is shown in SEQ ID NO: 46, and the amino acid sequence of chain 2 is shown in SEQ ID NO: 35.

In some embodiments, the bispecific antibody as described above includes two chains 1 with the same sequence and two chains 2 with the same sequence,

The chain 1 amino acid sequence is shown in SEQ ID NO: 44, and the chain 2 amino acid sequence is shown in SEQ ID NO: 31; or

The chain 1 amino acid sequence is shown in SEQ ID NO: 46, and the chain 2 amino acid sequence is shown in SEQ ID NO: 35.

In some embodiments, the bispecific antibody as described above, wherein the bispecific antibody has an IgG-scFv structure, and the anti-CD3 single chain antibody is linked to the C-terminus of the knob heavy chain or linked to the hole heavy chain C-terminal.

In some embodiments, the bispecific antibody as described above includes chain 1, chain 2, and chain 3, and the chain 1 comprises VH(CEA)-CH1-Fc(knob)-scFv( CD3), where VH(CEA)-CH1-Fc(knob) is fused to scFv(CD3) directly or through a linker;

The chain 2 includes VL(CEA)-CL from the amino terminus to the carboxy terminus; and

The chain 3 includes VH(CEA)-CH1-Fc(hole) from the amino terminus to the carboxy terminus; or

The chain 1 contains VH(CEA)-CH1-Fc(hole)-scFv(CD3) from the amino terminus to the carboxyl terminus, wherein VH(CEA)-CH1-Fc(hole) is fused to scFv(CD3) directly or through a linker ；

The chain 2 contains VL(CEA)-CL from the amino terminal to the carboxyl terminal; and the chain 3 contains VH(CEA)-CH1-Fc(knob) from the amino terminal to the carboxyl terminal; preferably,

(1) The amino acid sequence of the chain 1 is shown in SEQ ID NO: 28, or has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 28, and the amino acid sequence of the chain 3 is shown in SEQ ID NO ：43, or at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 43, and the amino acid sequence of chain 2 is shown in SEQ ID NO: 31, or with SEQ ID NO: 31 The amino acid sequence shown has at least 80% sequence identity; or

(m) The amino acid sequence of chain 1 is shown in SEQ ID NO: 32, or has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 32, and the amino acid sequence of chain 3 is shown in SEQ ID NO : 45, or at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 45, and the amino acid sequence of chain 2 as shown in SEQ ID NO: 35, or with SEQ ID NO: 35 The amino acid sequence shown has at least 80% sequence identity.

In some embodiments, the aforementioned (1) or (m) having at least 80% sequence identity is: having at least 80%, 82%, 84%, 85%, 86%, 88%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

In some embodiments, the bispecific antibody as described above includes chain 1, chain 2 and chain 3,

The amino acid sequence of the chain 1 is shown in SEQ ID NO: 28, the amino acid sequence of the chain 3 is shown in SEQ ID NO: 43, and the amino acid sequence of the chain 2 is shown in SEQ ID NO: 31; or

The amino acid sequence of chain 1 is shown in SEQ ID NO: 32, the amino acid sequence of chain 3 is shown in SEQ ID NO: 45, and the amino acid sequence of chain 2 is shown in SEQ ID NO: 35.

In some embodiments, the bispecific antibody as described above includes one chain 1, two chains 2 with the same sequence, and one chain 3.

The amino acid sequence of the chain 1 is shown in SEQ ID NO: 28, the amino acid sequence of the chain 3 is shown in SEQ ID NO: 43, and the amino acid sequence of the chain 2 is shown in SEQ ID NO: 31; or

The amino acid sequence of chain 1 is shown in SEQ ID NO: 32, the amino acid sequence of chain 3 is shown in SEQ ID NO: 45, and the amino acid sequence of chain 2 is shown in SEQ ID NO: 35.

The present disclosure also provides a nucleic acid molecule that encodes the bispecific antibody as described above.

In some embodiments, the nucleic acid molecule as described above comprises a sequence encoding each polypeptide chain of the bispecific antibody as described above.

In some embodiments, the nucleic acid molecule as described above comprises a nucleic acid sequence encoding one or more polypeptide chains selected from:

VH(CEA)-CH1-Fc-scFv(CD3), VL(CEA)-CL, VH(CEA)-CH1-Fc(knob)-scFv(CD3), VH(CEA)-CH1-Fc(hole), VH(CEA)-CH1-Fc(hole)-scFv(CD3) and VH(CEA)-CH1-Fc(knob).

The present disclosure also provides a host cell, which contains the nucleic acid molecule as described above.

In some embodiments, the host cell is a prokaryotic cell (E. coli, etc.) or a eukaryotic cell (yeast, mammalian cell, etc.). Preferably, the eukaryotic cell is a mammalian cell, including but not Limited to 293 cells, CHO cells, Sp2/0 cells, COS cells, HEK cells and NS0 cells. The mammalian cells mentioned herein do not include embryonic cells directly derived from humans.

The present disclosure also provides a pharmaceutical composition, which contains a therapeutically effective amount of the bispecific antibody as described above, or the nucleic acid molecule as described above, and one or more pharmaceutically acceptable carriers , Diluent, buffer or excipient; preferably, the unit dose of the pharmaceutical composition may contain 0.01 to 99% by weight of the bispecific antibody or the amount of the bispecific antibody in the unit dose of the pharmaceutical composition It is 0.1-3000 mg, more preferably 1-1000 mg.

The present disclosure also provides a method for treating diseases associated with CEA-positive cells, the method comprising administering to a subject a therapeutically effective amount of a bispecific antibody as described above, or a nucleic acid molecule as described above, or The pharmaceutical composition as described above, wherein the disease is preferably a CEA-positive tumor or cancer; more preferably, the disease is selected from: melanoma, bowel cancer, non-small cell lung cancer (NSCLC), gastric cancer, pancreatic cancer, Breast cancer, lung cancer, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, bladder cancer, gallbladder cancer, esophageal cancer and medullary thyroid cancer.

The present disclosure also provides the use of the aforementioned bispecific antibody, or the aforementioned nucleic acid molecule, or the aforementioned pharmaceutical composition in the preparation of a medicine for treating diseases associated with CEA-positive cells, more preferably The disease is selected from: melanoma, bowel cancer, non-small cell lung cancer (NSCLC), gastric cancer, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, bladder cancer, gallbladder cancer, Esophageal cancer and medullary thyroid cancer.

The present disclosure also provides the bispecific antibody as described above, or the nucleic acid molecule as described above, or the pharmaceutical composition as described above for use as a medicine. Preferably, the medicine is used for the treatment of CEA-positive cells. The disease is selected from: melanoma, bowel cancer, non-small cell lung cancer (NSCLC), gastric cancer, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, bladder cancer, gallbladder cancer, Esophageal cancer and medullary thyroid cancer.

The present disclosure also provides the use of the aforementioned bispecific antibody, or the aforementioned nucleic acid molecule, or the aforementioned pharmaceutical composition in the preparation of a medicine for the treatment of cancer; preferably, wherein the cancer is selected From: melanoma, bowel cancer, non-small cell lung cancer (NSCLC), gastric cancer, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, bladder cancer, gallbladder cancer, esophageal cancer and medullary thyroid Cancer; more preferably, wherein the cancer is a CEA positive cancer.

The present disclosure also provides a method for treating cancer, the method comprising administering to a subject a therapeutically effective amount of the bispecific antibody as described above, or the nucleic acid molecule as described above, or the drug combination as described above Preferably, wherein the cancer is selected from: melanoma, bowel cancer, non-small cell lung cancer (NSCLC), stomach cancer, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, bladder Cancer, gallbladder cancer, esophageal cancer and medullary thyroid cancer; more preferably, the cancer is CEA-positive cancer.

The present disclosure also provides the bispecific antibody as described above, or the nucleic acid molecule as described above, or the pharmaceutical composition as described above for use as a medicine for the treatment of cancer. Preferably, the cancer is selected from: melanoma , Bowel cancer, non-small cell lung cancer (NSCLC), stomach cancer, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, bladder cancer, gallbladder cancer, esophageal cancer and medullary thyroid cancer; more preferred Ground, wherein the cancer is a CEA positive cancer.

Description of the drawings

Figure 1: Flow cytometry to detect the binding of bispecific antibodies to granulocytes.

Figure 2A shows that hu63-18T, hu67-19B and hu67-18T can effectively activate Jurkat-Lucia ^TM NFAT cell line in the presence of CEA-positive cells LS174T; Figure 2B shows that in the presence of CEA-negative tumor cell line HCT116, each None of the bispecific antibodies can activate the Jurkat-Lucia ^™ NFAT cell line; Figure 2C shows that in the presence of tumor-free cell lines, none of the bispecific antibodies can activate the Jurkat-Lucia ^™ NFAT cell line.

Figure 3: Anti-tumor efficacy of bispecific antibody in mouse LS174T model reconstructed with human PBMC.

Detailed ways

Definition of Terms)

To make it easier to understand this disclosure, certain technical and scientific terms are specifically defined below. Unless clearly defined otherwise herein, all other technical and scientific terms used herein have meanings commonly understood by those of ordinary skill in the art to which this disclosure belongs.

The singular forms "a", "an" and "the" used in the specification and claims include plural references unless the context clearly indicates otherwise.

Unless the context clearly requires otherwise, throughout the specification and claims, the words "including", "having", "including", etc. should be understood as having an inclusive meaning rather than an exclusive or exhaustive meaning; that is, " Including but not limited to the meaning of ".

The terms "and/or", such as "X and/or Y" should be understood to mean "X and Y" or "X or Y" and should be used to provide clear support for both or either meaning.

The three-letter codes and one-letter codes of amino acids used in this disclosure are as described in J. biool. chem, 243, p3558 (1968).

The term "bispecific antibody" refers to a protein molecule that can specifically bind to two target antigens or target antigen epitopes, and contains antibodies or antigen-binding fragments of antibodies (such as single-chain antibodies). Among the bispecific antibodies capable of binding to the two targets of CEA and CD3, there are an antibody portion capable of anti-CEA and an antibody portion capable of anti-CD3. The "antibody portion" may be a full-length antibody or a full-length IgG antibody capable of specifically binding CEA or CD3, or a full-length antibody or an antigen-binding fragment of a full-length IgG antibody capable of specifically binding CEA or CD3. In some embodiments, the bispecific antibody described in the present disclosure has the structure of IgG-(scFv)2. In some embodiments, the bispecific antibody therein comprises two chains 1 with the same sequence and two chains 2 with the same sequence. In some embodiments, the bispecific antibodies described in the present disclosure have the structure of IgG-scFv. In some embodiments, the bispecific antibody includes 4 chains, including one chain 1, two chains 2 with the same sequence, and one chain 3.

The term "binding region" or "binding region" for an antigen refers to a region or part that can specifically bind to an antigen in a multispecific protein molecule or an antibody molecule. The antigen binding region can be a region or part that can directly bind to the antigen. The part of the ligand binding domain to be bound may also be a domain containing the variable region of an antibody that can directly bind to an antigen.

The "antibody" mentioned in the present disclosure refers to immunoglobulin. A natural antibody is usually a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different. According to this, immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE. The corresponding heavy chains are μ chain, δ chain, and γ chain. , Α chain, and ε chain. The same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chain is divided into a kappa chain or a lambda chain by the difference of the constant region. Each of the five types of Ig can have a kappa chain or a lambda chain.

The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly and is the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region. The variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conserved sequences. Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDR). Each light chain variable region (VL) and heavy chain variable region (VH) consists of 3 CDR regions and 4 FR regions. The sequence from the amino terminal to the carboxy terminal is: FR1, CDR1, FR2, CDR2 , FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

The antibodies of the present disclosure include murine antibodies, chimeric antibodies and humanized antibodies. In addition to full-length antibodies, the antibodies of the present disclosure also include antigen-binding fragments capable of binding antigen.

The term "murine antibody" in the present disclosure is a murine-derived monoclonal antibody against human CEA (or its epitope) prepared according to the knowledge and skills in the art. During the preparation, the test subject is injected with the CEA antigen (or its epitope), and then the hybridoma expressing the antibody with the desired sequence or functional characteristics is isolated. In a preferred embodiment of the present disclosure, the murine anti-CEA antibody or antigen-binding fragment thereof may further comprise the light chain constant region of murine kappa, lambda chains or variants thereof, or further comprise murine IgG1, The heavy chain constant region of IgG2, IgG3, or variants thereof.

The term "chimeric antibody" is an antibody formed by fusing the variable region of the antibody of the first species with the constant region of the antibody of the second species, which can alleviate the immune response induced by the heterologous antibody. For example, to establish a chimeric antibody, a hybridoma that secretes murine-specific monoclonal antibodies must be established first, and then the variable region genes from the murine hybridoma cells will be cloned, and then the constant region genes of the human antibody will be cloned as needed. The region gene and the human constant region gene are connected to form a chimeric gene and then inserted into an expression vector, and finally the chimeric antibody molecule is expressed in a eukaryotic system or a prokaryotic system. In a preferred embodiment of the present disclosure, the antibody light chain of the CEA chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or a variant thereof. The antibody heavy chain of the CEA chimeric antibody further comprises the heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or variants thereof, preferably comprises the human IgG1, IgG2 or IgG4 heavy chain constant region, or introduces amino acid mutations (E.g. L234A and/or L235A mutation, and/or S228P mutation) IgG1, IgG2 or IgG4 variants.

The term "humanized antibody" is also called CDR-grafted antibody, which refers to the transplantation of CDR sequences from non-human species to the framework of human antibody variable regions, that is, in different types of human species The antibody produced in the framework sequence of the antibody. Humanized antibodies can overcome the heterologous reaction induced by chimeric antibodies that carry a large amount of heterologous protein components. Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, the germline DNA sequences of the human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet www.mrccpe.com.ac.uk/vbase ), as well as in Kabat, EA, etc. Human, 1991 Sequences of Proteins of Immunological Interest, found in the 5th edition. In order to avoid the decrease of immunogenicity and the resulting decrease in activity, the human antibody variable region framework sequence can be subjected to minimal reverse mutations or back mutations to maintain activity. The humanized antibodies of the present disclosure also include humanized antibodies that have been further displayed by yeast to undergo affinity maturation mutations to the CDRs.

Due to the contact residues of the antigen, the grafting of the CDR may result in the reduced affinity of the produced antibody or its antigen-binding fragment to the antigen due to the framework residues in contact with the antigen. Such interactions may be the result of high mutations in somatic cells. Therefore, it may still be necessary to transplant such donor framework amino acids to the framework of the humanized antibody. The amino acid residues involved in antigen binding from non-human antibodies or antigen-binding fragments thereof can be identified by examining the sequence and structure of the variable region of animal monoclonal antibodies. Residues in the CDR donor framework that differ from the germline can be considered related. If the closest germline cannot be determined, the sequence can be compared with the consensus sequence of a subclass or animal antibody sequence with a high percentage of similarity. Rare framework residues are thought to be the result of somatic hypermutation and thus play an important role in binding.

In one embodiment of the present disclosure, the antibody or antigen-binding fragment thereof may further comprise a light chain constant region of human or murine κ, λ chain or a variant thereof, or further comprise human or murine IgG1, The heavy chain constant region of IgG2, IgG3, IgG4 or variants thereof; preferably comprising human IgG1, IgG2, or IgG4 heavy chain constant region, or IgG1 with amino acid mutations (such as L234A and/or L235A mutations, and/or S228P mutations) introduced , IgG2 or IgG4 variants.

The "conventional variants" of the human antibody heavy chain constant region and the human antibody light chain constant region described in the present disclosure refer to the heavy chain constant region that has been disclosed in the prior art and does not change the structure and function of the antibody variable region. Or variants of the constant region of the light chain. Exemplary variants include IgG1, IgG2, IgG3 or IgG4 heavy chain constant region variants with site-directed modification of the heavy chain constant region and amino acid substitutions. The specific substitutions are YTE known in the art. Mutations, L234A and/or L235A mutations, S228P mutations, and/or mutations to obtain a knob-into-hole structure (making the antibody heavy chain have a combination of knob-Fc and hole-Fc), these mutations have been confirmed to make the antibody have novel Performance, but does not change the function of the antibody variable region.

"HuMAb" (HuMAb), "human antibody", "fully human antibody", and "fully human antibody" can be used interchangeably, and can be antibodies derived from humans or antibodies obtained from a genetically modified organism. The transgenic organism is "engineered" to produce specific human antibodies in response to antigen stimulation and can be produced by any method known in the art. In some technologies, the elements of human heavy and light chain loci are introduced into cell lines of organisms derived from embryonic stem cell lines, and the endogenous heavy and light chain loci in these cell lines are targeted. To destruction. Transgenic organisms can synthesize human antibodies specific to human antigens, and the organisms can be used to produce human antibody-secreting hybridomas. A human antibody can also be an antibody in which the heavy and light chains are encoded by nucleotide sequences derived from one or more human DNA sources. Fully human antibodies can also be constructed by gene or chromosome transfection methods and phage display technology, or constructed from B cells activated in vitro, all of which are known in the art.

The terms "full-length antibody", "whole antibody", "whole antibody" and "whole antibody" are used interchangeably herein and refer to an antibody in a substantially complete form, as distinguished from the antigen-binding fragments defined below. The term specifically refers to antibodies whose light and heavy chains contain constant regions. The "antibody" of the present disclosure includes "full-length antibodies" and antigen-binding fragments thereof.

The term "IgG-(scFv)2 structure" refers to a bispecific antibody structure assembled from two heavy chains with the VHa-CH1-Fc-scFvb structure and two light chains with the same sequence of the VLa-CL structure, Among them, VHa and VLa form an antigen-binding fragment that binds to antigen a or epitope a, and scFvb is a single-chain antibody that binds antigen b or epitope b. The Fc sequence of the two heavy chains may be the same or different.

The term "IgG-scFv structure" refers to a heavy chain with a VHa-CH1-Fc(knob)-scFvb structure (called a knob heavy chain), and a heavy chain with a VHa-CH1-Fc(hole) structure (called a hole). Heavy chain) and two VLa-CL structural light chains with the same sequence as a bispecific antibody structure; or a heavy chain with a VHa-CH1-Fc (knob) structure (called a knob heavy chain), a A bispecific antibody structure composed of a heavy chain of VHa-CH1-Fc(hole)-scFvb structure (called hole heavy chain) and two light chains of the same sequence of VLa-CL structure, where VHa and VLa form a binding a antigen Or an antigen-binding fragment of a epitope, scFvb is a single-chain antibody that binds to the b antigen or the b epitope.

In some embodiments, the full-length antibody of the present disclosure includes a light chain in which a light chain variable region is connected to a light chain constant region, and a full-length antibody formed by a heavy chain in which a heavy chain variable region is connected to a heavy chain constant region. Those skilled in the art can select different antibody-derived light chain constant regions and heavy chain constant regions according to actual needs, for example, human antibody-derived light chain constant regions and heavy chain constant regions.

The term "antigen-binding fragment" of an antibody refers to one or more fragments of the antibody that retain the ability to specifically bind to an antigen (eg, CEA) or an epitope thereof. It has been shown that fragments of full-length antibodies can be used to achieve the antigen-binding function of antibodies. Examples of the binding fragment contained in the term "antigen-binding fragment" of the antibody include (i) Fab fragments, which are monovalent fragments composed of VL, VH, CL and CH1 domains; (ii) F(ab') ₂ fragments, It is a bivalent fragment comprising two Fab fragments connected by a disulfide bridge on the hinge region; (iii) Fd fragment composed of VH and CH1 domains; (iv) VH and VL domains of one arm of an antibody (V) dsFv, which is a stable antigen-binding fragment formed by interchain disulfide bonds between VH and VL; (vi) diabodies, bispecific antibodies and other fragments including scFv, dsFv, Fab, etc. Multispecific antibodies. In addition, although the two domains VL and VH of the Fv fragment are encoded by separate genes, recombination methods can be used to connect them through a synthetic linker so that it can be produced as a single protein in which the VL and VH regions are paired to form a monovalent molecule. Chain (referred to as single chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85: 5879-5883). Such single-chain antibodies are also included in the term "antigen-binding fragments" of antibodies. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as for intact antibodies. The antigen binding portion can be produced by recombinant DNA technology or by enzymatic or chemical fragmentation of the intact immunoglobulin. The antibodies may be antibodies of different isotypes, for example, IgG (eg, IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.

Fab is an antibody fragment with a molecular weight of about 50,000 among the fragments obtained by treating IgG antibody molecules with enzymes, and an antibody fragment with antigen-binding activity, in which about half of the N-terminal side of the heavy chain and the entire L chain are bonded by disulfide bonds Together.

F(ab')2 is a fragment obtained by enzymatically digesting the downstream part of two disulfide bonds in the hinge region of IgG. It has a molecular weight of about 100,000 and has antigen binding activity. It contains two Fabs connected at the hinge position. Area.

Fab' is a fragment obtained by cleaving the disulfide bond in the hinge region of F(ab')2, and has antigen-binding activity. The Fab' of the present disclosure can be produced by treating F(ab') 2 of the present disclosure with a reducing agent.

In addition, the Fab' can be produced by inserting DNA encoding the Fab' fragment of the antibody into a prokaryotic expression vector or a eukaryotic expression vector, and introducing the vector into a prokaryotic organism or eukaryotic organism to express Fab'.

The term "single-chain antibody", "single-chain Fv" or "scFv" means to comprise an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker Of molecules. Such scFv molecules may have the general structure: NH ₂ -VL-linker-VH-COOH or NH _{2 -VH-linker-VL-COOH.} Suitable prior art linkers consist of a repeated GGGGS amino acid sequence or variants thereof, for example using 1-4 repeated variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448) . Other linkers that can be used in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996) , Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.

Diabodies are antibody fragments with bivalent antigen binding activity. In the bivalent antigen binding activity, the two antigens can be the same or different.

Bispecific antibodies and multispecific antibodies refer to antibodies that can simultaneously bind to two or more antigens or antigenic determinants, including scFv or Fab fragments that can bind CEA and CD3.

dsFv can be obtained, for example, by substituting one amino acid residue in VH and VL with a cysteine residue, and a fragment obtained by forming a disulfide bond between the cysteine residues. The amino acid residues substituted with cysteine residues can be selected based on the prediction of the three-dimensional structure of the antibody according to a known method (Protein Engineering, 7, 697 (1994)).

The term "amino acid difference" or "amino acid mutation" means that compared with the original protein or polypeptide, the variant protein or polypeptide has amino acid changes or mutations, including one, two, three, or mutations on the basis of the original protein or polypeptide. Insertion, deletion or substitution of more amino acids.

The term "antibody framework" or "FR region" refers to a part of the variable domain VL or VH, which serves as a scaffold for the antigen binding loop (CDR) of the variable domain. Essentially, it is a variable domain without CDRs.

The term "complementarity determining region", "CDR" or "hypervariable region" refers to one of the six hypervariable regions in the variable domain of an antibody that mainly contribute to antigen binding. Generally, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region, and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region. Any one of various well-known schemes can be used to determine the amino acid sequence boundaries of CDRs, including the "Kabat" numbering rule (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), "Chothia" numbering rules (Martin, ACR.Protein Sequence and Structure Analysis of Antibody Variable Domains[J].2001) and ImmunoGenTics (IMGT) numbering rules (Lefranc, MP, etc., Dev. Comp. Immunol., 27, 55-77 (2003), etc. For example, for the classical format, following the Kabat rule, the CDR amino acid residues in the variable domain of the heavy chain (VH) are numbered 31-35 (HCDR1) , 50-65 (HCDR2) and 95-102 (HCDR3); CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 ( LCDR3). Following the Chothia rule, the CDR amino acid numbers in VH are 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3); and the amino acid residues in VL are numbered 24-34 (LCDR1) , 50-56 (LCDR2) and 89-97 (LCDR3). Following IMGT rules, the CDR amino acid residue numbers in VH are roughly 27-38 (CDR1), 56-65 (CDR2) and 105-117 (CDR3), The CDR amino acid residue numbers in VL are roughly 27-38 (CDR1), 56-65 (CDR2) and 105-117 (CDR3). Following IMGT rules, the CDR regions of antibodies can be determined using the program IMGT/DomainGap Align.

The term "epitope" or "antigenic determinant" refers to a site on an antigen that is specifically bound by an immunoglobulin or antibody (for example, a specific site on a CEA molecule). Epitopes usually include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-contiguous amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular B iology, Volume 66, G.E. Morris, Ed. (1996).

The terms "specifically binds", "selectively binds", "selectively binds" and "specifically binds" refer to the binding of an antibody to an epitope on a predetermined antigen. Generally, antibodies bind with an affinity (KD) of ^{about less than 10 -7} M, for example, about less than 10 ^-8 M, 10 ^-9 M, 10 ^-10 M, 10 ^-11 M, 10 ^{-12 M or less.}

The term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. For example, in the present disclosure, the affinity of the antibody and the cell surface antigen is determined by the FACS method or Biacore to determine the KD value.

The term "nucleic acid molecule" as used herein refers to DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, and is preferably double-stranded DNA or single-stranded mRNA or modified mRNA. When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, the nucleic acid is "operably linked." For example, if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is effectively linked to the coding sequence.

Amino acid sequence "identity" means that when aligning amino acid sequences, gaps are introduced when necessary to achieve the maximum sequence identity percentage, and any conservative substitutions are not regarded as part of sequence identity. The first sequence is compared with the second sequence. The percentage of amino acid residues in which the amino acid residues are identical. For the purpose of determining the percentage of amino acid sequence identity, the alignment can be achieved in a variety of ways within the technical scope of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring the alignment, including any algorithms required to achieve the maximum alignment over the entire length of the sequence being compared.

The term "expression vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. In another embodiment, the vector is a viral vector in which additional DNA segments can be ligated into the viral genome. The vectors disclosed herein can replicate autonomously in the host cell into which they have been introduced (for example, bacterial vectors with a bacterial origin of replication and episomal mammalian vectors) or can be integrated into the genome of the host cell after being introduced into the host cell, so as to follow The host genome replicates together (e.g., a non-episomal mammalian vector).

The methods for producing and purifying antibodies and antigen-binding fragments are well known in the prior art, such as Cold Spring Harbor’s Antibody Experiment Technical Guide, Chapters 5-8 and 15. For example, mice can be immunized with human CEA or fragments thereof, and the obtained antibodies can be renatured, purified, and amino acid sequencing can be performed by conventional methods. Antigen-binding fragments can also be prepared by conventional methods. The antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions to the non-human CDR regions. The human FR germline sequence can be obtained from the ImmunoGeneTics (IMGT) website http://imgt.cines.fr by comparing the IMGT human antibody variable region germline gene database and MOE software, or from the Journal of Immunoglobulin, 2001ISBN012441351 obtain.

The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacteria, microorganisms, plant or animal cells. Bacteria that are easily transformed include members of the enterobacteriaceae, such as Escherichia coli or Salmonella strains; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese Hamster Ovary cell line), 293 cells and NSO cells.

The engineered bispecific antibodies of the present disclosure can be prepared and purified using conventional methods. For example, the cDNA sequence encoding each chain of the bispecific antibody can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As a more recommended prior art, mammalian expression systems can lead to glycosylation of bispecific antibodies, especially in the highly conserved N-terminal sites of the Fc region. Stable clones are obtained by expressing bispecific antibodies that specifically bind to human CEA and CD3. Positive clones are expanded in the serum-free medium of the bioreactor to produce antibodies. The culture medium from which the antibody is secreted can be purified by conventional techniques. For example, use A or G Sepharose FF column with adjusted buffer for purification. Wash away non-specifically bound components. Then the bound antibody was eluted by the pH gradient method, and the antibody fragment was detected by SDS-PAGE and collected. The antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The resulting product needs to be frozen immediately, such as -70°C, or lyophilized.

"Administration", "administration" and "treatment" when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids refer to exogenous drugs, therapeutic agents, diagnostic agents or compositions and animals , Human, subject, cell, tissue, organ or biological fluid contact. "Administration", "administration" and "treatment" can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods. The treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, where the fluids are in contact with cells. "Administration", "administration" and "treatment" also mean the treatment of, for example, cells by reagents, diagnostics, binding compositions, or by another cell in vitro and ex vivo. "Treatment" when applied to human, veterinary or research subjects, refers to treatment, preventive or preventive measures, research and diagnostic applications.

"Treatment" means administering an internal or external therapeutic agent to a subject, for example, a composition comprising any one of the bispecific antibodies of the present disclosure, the subject has one or more disease symptoms, and the subject is known to The therapeutic agent has a therapeutic effect on these symptoms. Generally, the therapeutic agent is administered to the subject or population to be treated in an amount effective to relieve one or more symptoms of the disease, in order to induce the regression of such symptoms or inhibit the development of such symptoms to any clinically measurable degree. The amount of the therapeutic agent effective to alleviate the symptoms of any particular disease (also referred to as a "therapeutically effective amount") can vary depending on various factors, such as the subject’s disease state, age and weight, and the amount of the drug that produces the desired therapeutic effect in the subject ability. Any clinical testing methods commonly used by doctors or other professional health care professionals to evaluate the severity or progression of the symptoms can evaluate whether the symptoms of the disease have been alleviated. Although the embodiments of the present disclosure (such as treatment methods or products) may be ineffective in alleviating the symptoms of each target disease, according to any statistical test methods known in the art such as Student's t test, chi-square test, Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should reduce the symptoms of the target disease in a statistically significant number of subjects.

"Conservative modification" or "conservative substitution or substitution" means that other amino acids with similar characteristics (such as charge, side chain size, hydrophobicity/hydrophilicity, main chain conformation and rigidity, etc.) replace amino acids in a protein so that they can be frequently Make changes without changing the biological activity of the protein. Those skilled in the art know that, generally speaking, a single amino acid substitution in a non-essential region of a polypeptide does not substantially change the biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., Page 224, (4th edition)). In addition, the substitution of amino acids with similar structure or function is unlikely to disrupt biological activity. Exemplary conservative substitutions are set out in the table "Exemplary Amino Acid Conservative Substitutions" below.

Exemplary amino acid conservative substitutions

Original residue	Conservative substitution
Ala(A)	Gly; Ser
Arg(R)	Lys; His
Asn(N)	Gln; His; Asp
Asp(D)	Glu; Asn
Cys(C)	Ser; Ala; Val
Gln(Q)	Asn; Glu
Glu(E)	Asp; Gln
Gly(G)	Ala
His(H)	Asn; Gln
Ile(I)	Leu; Val
Leu(L)	Ile; Val
Lys(K)	Arg; His
Met(M)	Leu; Ile; Tyr
Phe(F)	Tyr; Met; Leu
Pro(P)	Ala
Ser(S)	Thr
Thr(T)	Ser
Trp(W)	Tyr; Phe
Tyr(Y)	Trp; Phe
Val(V)	Ile; Leu

"Effective amount" or "effective dose" refers to the amount of the drug, compound or pharmaceutical composition necessary to obtain any one or more beneficial or desired preventive/therapeutic results. For prophylactic use, beneficial or desired results include elimination or reduction of risk, reduction of severity, or delay of the onset of the disease, including the biochemistry, tissue, and organization of the disease, its complications, and intermediate pathological phenotypes that appear during the development of the disease. Academic and/or behavioral symptoms. For therapeutic applications, beneficial or desired results include clinical results, such as reducing the incidence of various target antigen-related disorders of the present disclosure or improving one or more symptoms of the disorder, and reducing the effectiveness of other agents required to treat the disorder. Dosage, enhance the efficacy of another agent, and/or delay the progression of the patient’s disease related to the target antigen of the present disclosure.

"Exogenous" refers to substances produced outside organisms, cells, or humans according to circumstances. "Endogenous" refers to substances that are produced in cells, organisms, or humans depending on the situation.

"Homology" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in the two comparison sequences are occupied by the same base or amino acid monomer subunit, for example, if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position . The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100. For example, in the optimal sequence alignment, if there are 6 matches or homology in 10 positions in the two sequences, then the two sequences are 60% homologous; if there are 95 matches in 100 positions in the two sequences Or homology, then the two sequences are 95% homologous. Generally, when two sequences are aligned, a comparison is made to give the maximum percentage homology. For example, the comparison can be performed by the BLAST algorithm, in which the parameters of the algorithm are selected to give the maximum match between each sequence over the entire length of each reference sequence. The following references refer to the BLAST algorithm frequently used for sequence analysis: BLAST algorithm (BLAST ALGORITHMS): Altschul, SF et al., (1990) J. Mol. Biol. 215:403-410; Gish, W. et al., (1993) ) Nature Genet. 3:266-272; Madden, TL et al., (1996) Meth. Enzymol. 266:131-141; Altschul, SF et al., (1997) Nucleic Acids Res. 25: 3389-3402; Zhang, J. et al. (1997) Genome Res. 7:649-656. Other conventional BLAST algorithms provided by NCBI BLAST are also well known to those skilled in the art.

As used herein, the expressions "cell", "cell line" and "cell culture" are used interchangeably, and all such names include progeny. Therefore, the words "transformant" and "transformed cell" include primary test cells and cultures derived therefrom, regardless of the number of passages. It should also be understood that due to deliberate or unintentional mutations, all offspring cannot be exactly the same in terms of DNA content. Including mutant progeny with the same function or biological activity as screened in the original transformed cell. Where a different name is meant, it is clearly visible from the context.

"Optional" or "optionally" means that the event or environment described later can but does not have to occur, and the description includes occasions where the event or environment occurs or does not occur.

"Pharmaceutical composition" means a mixture containing one or more of the bispecific antibodies described herein, or their physiologically/pharmaceutically acceptable salts or prodrugs, and other chemical components; the other Components such as physiological/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredient and thus the biological activity.

The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in a formulation for the delivery of antibodies or antigen-binding fragments. The carrier can be an anti-adhesive agent, binder, coating, disintegrant, filler or diluent, preservative (such as antioxidant, antibacterial or antifungal), sweetener, absorption delaying agent, wetting agent Agent, emulsifier, buffer, etc. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), dextrose, vegetable oils (e.g. olive oil), saline, buffers, buffered saline, and the like Penetrating agents such as sugars, polyols, sorbitol, and sodium chloride.

In addition, the present disclosure includes agents for treating diseases associated with target antigen (for example, CEA) positive cells, the agents comprising the anti-CEA antibody or bispecific antibody of the present disclosure as an active ingredient.

The disease related to CEA-positive cells in the present disclosure is not limited, as long as it is a disease related to CEA-positive cells. For example, the therapeutic response induced by the molecule of the present disclosure can be achieved by binding to human CEA and then blocking CEA and its receptor/coordination Binding to the body, or kill the tumor cells that overexpress CEA. Therefore, when in preparations and preparations suitable for therapeutic applications, the bispecific antibodies of the present disclosure are very useful for people who have tumors or cancers, preferably melanoma, bowel cancer, and non-small cell lung cancer. (NSCLC), stomach cancer, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, bladder cancer, gallbladder cancer, esophageal cancer, medullary thyroid cancer, etc.

In addition, the present disclosure relates to methods for immunodetection or determination of target antigens (for example, CEA), reagents for immunodetection or determination of target antigens (for example, CEA), and for immunodetection or determination of cells expressing target antigens (for example, CEA) The method and the diagnostic agent for diagnosing diseases related to the target antigen (for example, CEA) positive cell, which comprises the disclosed specific recognition target antigen (for example, human CEA) and combined with the amino acid sequence of the extracellular region or its three-dimensional structure The bispecific antibody is used as the active ingredient.

In the present disclosure, the method for detecting or measuring the amount of the target antigen (for example, CEA) may be any known method. For example, it includes immunodetection or assay methods.

The immunodetection or measurement method is a method of detecting or measuring the amount of antibody or antigen using a labeled antigen or antibody. Examples of immunodetection or measurement methods include radioactive substance-labeled immunoantibody method (RIA), enzyme immunoassay (EIA or ELISA), fluorescent immunoassay (FIA), luminescence immunoassay, western blotting, physicochemical method Wait.

The above-mentioned diseases related to CEA-positive cells can be diagnosed by detecting or measuring CEA-expressing cells with the bispecific antibody of the present disclosure.

In order to detect cells expressing polypeptides, known immunodetection methods can be used, and immunoprecipitation, fluorescent cell staining, immunotissue staining, and the like are preferably used. In addition, a fluorescent antibody staining method using the FMAT8100HTS system (Applied Biosystem) can be used.

In the present disclosure, there is no particular limitation on the living body sample used to detect or measure the target antigen (for example, CEA), as long as it has the possibility of containing cells expressing the target antigen (for example, CEA), such as tissue cells, blood, plasma , Serum, pancreatic juice, urine, stool, tissue fluid or culture fluid.

According to the required diagnostic method, the diagnostic agent containing the monoclonal antibody or antibody fragment thereof of the present disclosure may also contain a reagent for performing an antigen-antibody reaction or a reagent for detecting a reaction. The reagents used to perform the antigen-antibody reaction include buffers, salts, and the like. The reagents used for detection include reagents commonly used in immunological detection or measurement methods, such as a labeled second antibody that recognizes the monoclonal antibody, its antibody fragment or its conjugate, and a substrate corresponding to the label.

The details of one or more embodiments of the present disclosure are set forth in the above specification. Although any methods and materials similar or identical to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described below. Through the specification and claims, other features, purposes and advantages of this disclosure will be obvious. In the specification and claims, unless the context clearly indicates otherwise, the singular form includes the plural referent. Unless otherwise defined, all technical and scientific terms used herein have ordinary meanings as understood by those of ordinary skill in the art to which this disclosure belongs. All patents and publications cited in the specification are incorporated by reference. The following examples are proposed to more fully illustrate the preferred implementation of the present disclosure. These embodiments should not be construed as limiting the scope of this disclosure in any way.

Example

Example 1: Preparation of CEA recombinant protein and stably transfected cells

1. Recombinant CEA antigen and the sequence of CEA protein expressed on the cell surface

The human CEA protein sequences encoding Fc and His tags were cloned into mammalian cell expression vectors, and expressed and purified in 293E cells to obtain recombinant proteins, which were used in the experiments of subsequent examples. At the same time, the untagged human CEA gene, human CEACAM1 gene and monkey CEA gene were transfected into CHO cells to form a CHO cell line expressing CEA protein on the cell surface for subsequent antibody screening and identification. The amino acid sequence of the related protein is as follows:

1. Human CEA-his (hCEA-His) protein sequence:

2. Human CEA-Fc (hCEA-Fc) protein sequence:

3. Monkey CEA-His (cynoCEA-His) protein sequence:

4. Human CEA (hCEA-CHO) protein sequence expressed on the surface of CHO cells:

5. Monkey CEA (cynoCEA-CHO) protein sequence expressed on the surface of CHO cells:

6. Human CEACAM1 (CEACAM1-CHO) protein sequence expressed on the surface of CHO cells:

2. Purification of related proteins

1. Purification of His-tagged protein

Centrifuge the cell expression supernatant sample at high speed to remove impurities, equilibrate the nickel column with PBS buffer (pH 7.4), wash 2-5 times the column volume, and apply the supernatant sample to the Ni Sepharose excel column at a certain flow rate. Wash the column with PBS buffer until the A ₂₈₀ reading drops to baseline, then wash the column with PBS+10mM imidazole to remove non-specifically bound contaminants, and collect the effluent, and finally elution with PBS solution containing 300mM imidazole. Protein and collect the elution peaks. After the collected eluate is concentrated, the sample buffer is replaced with a PBS solution with a desalting column for use in subsequent experiments.

2. Purification of Fc-containing proteins, chimeric antibodies and hybridoma antibodies

The cell expression supernatant sample was centrifuged at high speed to remove impurities, the Fc-containing recombinant protein and chimeric antibody expression supernatant were purified with a Protein A column, and the hybridoma expression supernatant was purified with a Protein G column. The supernatant is applied to the column at a certain flow rate. Rinse the column with PBS until the A280 reading drops to baseline. The target protein was eluted with 100 mM acetic acid at pH 3.0, and neutralized with 1M Tris-HCl at pH 8.0. The eluted sample is concentrated and replaced with PBS and then divided into equipment.

Example 2: Preparation of mouse anti-human CEA monoclonal antibody

1. Immunization and fusion

Mouse immunization uses hCEA-His protein and cyno-CEA-His protein, or hCEA-CHO cells and cynoCEA-CHO cells for cross-immunization. The amount of protein-immunized 50 ug for the first immunization, after immunization with 25 ug, immune cells for each 10 ⁷ cells / mouse, immunized once every two weeks. After 3 times of immunization, blood was taken to determine the titer of the antibody in the serum. The mice with high antibody titer in the serum and the titer tending to the plateau were selected for spleen cell fusion, and the splenic lymphocytes and myeloma cells were fused with a PEG-mediated fusion step Sp2/0 cells (

CRL-8287 ^TM ) was fused to obtain hybridoma cells. The fused hybridoma cells use MC semi-solid complete medium (RPMI-1640 medium containing 20% FBS, 1×HAT, 1×OPI and 2% Methyl cellulose) at a density of ^{0.5-1×10 6 cells/mL} Resuspend and aliquot into 35mm cell culture dishes and incubate at 37°C and 5% CO ₂ for 7-9 days. On 7-9 days after fusion, according to the size of cell clones, pick single cell clones into 200μL/well HT complete medium (RPMI-1640 medium containing 20% FBS, 1×HT and 1×OPI) In a 96-well cell culture plate, cultured at 37°C and 5% CO _{2 for} 3 days for detection.

2. Hybridoma cell screening

The preliminary screening of antibodies was carried out by enzyme-linked immunosorbent assay (ELISA) based on cell surface antigens. Spread the cells on an Elisa plate (Corning, Cat#3599) and incubate overnight in a 37°C incubator. When the cells are completely attached to the wall and the whole well is about to grow, remove the supernatant, wash once with PBS, and add cell fixative (Beyotime, Cat#P0098), place at room temperature for 45 minutes. Remove the fixing solution, wash the plate with a plate washer 3 times, add 5% skimmed milk powder, and seal at 37°C for more than 3 hours. Remove the blocking solution and wash the plates 3 times with the plate washer. The sealed cell plate can be stored at -20°C or used directly. When using, add gradient dilution of hybridoma cell culture supernatant, incubate at 37°C for 1h, wash the plate 3 times, add 100μL 10000 times diluted Goat anti-mouse IgG H&L (HRP) secondary antibody (Abcam, Cat#ab205719) , Incubate at 37°C for 1h, and wash the plate 3 times with a washing machine. Add 100μL of TMB (KPL, Cat#5120-0077) and place it at 37°C for 10min, add 100μL of 1M sulfuric acid to stop the reaction, and read the absorbance at 450nm with a microplate reader. When the test antibody binds to CEA on the cell surface without being competed by soluble CEA (sCEA), incubate the antibody with sCEA for 30 minutes before adding it to the cell plate.

The screened positive clones are expanded and cryopreserved and subcloned two to three times until a single-cell clone is obtained. The selected hybridoma clones are further prepared and purified by using a serum-free cell culture method. The obtained hybridoma antibody was detected by flow cytometry for the binding of the antibody to the CEA protein on the cell surface (see Test Example 1 of this disclosure for the method), and hybridoma cell lines with good binding activity were selected. Among them, the binding activity test results of monoclonal hybridoma cell lines mAb63 and mAb67 are shown in Table 1.

Table 1: Experimental results of binding of mouse antibodies to CEA protein on the cell surface

3. Determination of hybridoma antibody sequence

The monoclonal hybridoma cell lines mAb63 and mAb67 were selected to clone the sequence of the monoclonal antibody. The process is as follows: the logarithmic growth phase hybridoma cells are collected, RNA is extracted with Trizol (Invitrogen, Cat#15596-018), and reverse transcribed into cDNA. After PCR amplification using cDNA as a template, it was sent to a sequencing company for sequencing. The amino acid sequence of the antibody corresponding to the obtained DNA sequence is shown in Table 2 below.

Table 2: CDR sequences of anti-CEA antibodies

Remarks: The antibody CDR sequences in the table are determined according to the Kabat numbering system.

Example 3: Humanization of murine anti-human CEA monoclonal antibody

Humanized antibodies were prepared by conventional antibody humanization methods to obtain humanized antibodies of mAb63 and mAb67. In addition, an amino acid mutation was introduced into the HCDR2 of the mAb63 humanized antibody. The sequence of the mutated HCDR2 is: DIFPKSGNTDYNRKFKD (SEQ ID NO: 19, the underlined amino acid is the mutated amino acid). The mutation of a single amino acid in HCDR2 does not Will not affect the activity of mAb63 humanized antibody. The humanized light and heavy chain variable region sequences are shown in Table 3 below:

Table 3 Light and heavy chain variable regions of humanized anti-CEA antibodies

Preparation of humanized antibodies

The expression vectors for the light chain and heavy chain of the antibody are constructed separately, the humanized antibody light/heavy chains are respectively cross-paired and combined, and the 293E cells are transfected, and the culture supernatant is collected and purified to obtain the humanized full-length antibody. The constant region of the humanized antibody heavy chain can be selected from constant regions of IgG1, IgG2, IgG3, IgG4 and variants thereof. Exemplarily, the following human heavy chain IgG1 constant region variant 1 (as shown in SEQ ID NO: 24) or the following human IgG1 heavy chain constant region knob variants with a knob and hole structure respectively (as shown in SEQ ID NO: 25) and a human IgG1 heavy chain constant region hole variant (shown in SEQ ID NO: 26) is connected with the aforementioned humanized heavy chain variable region to form an antibody full-length heavy chain; humanized antibody light chain constant region It can be selected from constant regions selected from human kappa, lambda chains or variants thereof. Exemplarily, a human light chain constant region kappa chain (shown in SEQ ID NO: 27) is connected to the aforementioned humanized light chain variable region to form an antibody full-length light chain.

The sequence of the constant region variants of an exemplary antibody is as follows:

Human IgG1 heavy chain constant region variant 1: (SEQ ID NO: 24)

Human IgG1 heavy chain constant region knob variant: (SEQ ID NO: 25)

Human IgG1 heavy chain constant region hole variant: (SEQ ID NO: 26)

Human light chain constant region κ chain: (SEQ ID NO: 27)

The light chain and heavy chain variable regions of different humanized antibodies are respectively connected with different types of human light chain constant regions and human heavy chain constant regions to form humanized antibody light chains and heavy chains. The anti-CEA antibody sequence and the CD3 single chain antibody sequence are shown in Table 4 below.

Table 4. Humanized antibody light/heavy chain sequence and CD3scFv

Example 4. Construction of bispecific antibodies

The CEA specific antibody hu67-18, hu63-18, hu67-19 or hu63-19 and CD3 specific antibody (CD3scFv) were constructed into the form of IgG-(scFv) ₁ or IgG-(scFv) ₂ by bridge PCR, so The specific molecules (hu63-18T, hu67-18T, hu63-19B, hu67-19B) and sequences that form the bispecific antibody are as follows:

hu63-18T:

hu63-18T chain 1: (SEQ ID NO: 28)

hu63-18T chain 2: (SEQ ID NO: 31)

hu63-18T chain 3: (SEQ ID NO: 43)

hu63-19B

hu63-19B chain 1: (SEQ ID NO: 44)

hu63-19B chain 2: (SEQ ID NO: 31)

hu67-18T

hu67-18T chain 1 (SEQ ID NO: 32)

hu67-18T chain 2 (SEQ ID NO: 35)

hu67-18T chain 3 (SEQ ID NO: 45)

hu67-19B:

hu67-19B chain 1 (SEQ ID NO: 46)

hu67-19B chain 2 (SEQ ID NO: 35)

The amino acid sequence of the positive control molecule is as follows:

RG7802 (from patent application WO2017055389)

>RG7802 chain 1 (SEQ ID NO: 47)

>RG7802 chain 2 (SEQ ID NO: 48)

>RG7802 chain 3 (SEQ ID NO: 49)

>RG7802 chain 4 (SEQ ID NO: 50)

Test case

Test example 1. BIAcore detects the affinity of bispecific antibodies to CEA and CD3

A CM5 biosensor chip coupled with anti-human Fc antibody was used to affinity capture IgG, and then the antigen was flowed across the chip surface, and the reaction signal was detected in real time with a Biacore T200 instrument to obtain binding and dissociation curves. After the dissociation of each experimental cycle is completed, the biosensor chip is washed and regenerated _{with 3M MgCl 2.} The data fitting model adopts 1:1 Model.

Table 5. Biacore test results of the affinity of bispecific antibodies to CEA and CD3 antigens

Test Example 2: Determination of antibody binding capacity at the cellular level

The FACS method was used to detect the ability of bispecific antibodies to bind to cell surface antigens. The binding of the cell surface antigens CD3 and CEA was tested with Jurkat cells (ATCC, PTS-TIB-152) and HPAFII cells (ATCC, CRL-1997), respectively.

^{Add cells (2×10 6} cells/mL, 90 μL) resuspended in FACS buffer (98% PBS, 2% FBS) to a 96-well U-shaped bottom plate (Corning, 3795), and add 10 μL of serially diluted antibodies, 4 Incubate for 1 hour at ℃, wash with FACS buffer twice, then add APC anti-human-IgG Fc antibody (biolegend, Cat#409306, 1:200 dilution) to each well, incubate at 4°C for 30 minutes, wash twice, and regenerate with FACS buffer. Suspend the cells, and finally read the fluorescence signal value with FACS CantoII (BD).

Binding to Jurkat cells

hu67-18T and hu63-18T have the weakest binding to Jurkat cells, with EC50 of 225 and 275.5nM, respectively. Because of the two CD3 binding domains, hu67-19B has the strongest binding ability to Jurkat, with EC50 of 15.1nM.

Combines with HAPFII cells

The binding ability of hu67-18T, hu67-19B and hu63-18T to HPAFII cells is similar, with EC50 of 3.25, 4.92 and 3.52nM, respectively.

Table 6. FACS binding EC50 of bispecific antibodies to Jurkat cell line

Bispecific antibody	Jurkat cells (nM)	HAPFII cells (nM)
hu67-18T	225	3.25
hu67-19B	15.1	4.92
hu63-18T	275.5	3.52

Test Example 3. Binding of bispecific antibodies to granulocytes (granulocytes)

According to reports, there are endogenous expressions of CEACAM1, CEACAM6, and CEACAM8 on the surface of granulocytes. Therefore, it is possible to determine whether the antibody has cross-binding activity with other CEACAM family members CEACAM1, CEACAM6 and CEACAM8 by detecting whether the antibody binds to granulocytes.

After collecting fresh human peripheral blood, separate granulocytes according to the instructions of Ficoll Paque Premium 1.084, and perform density gradient centrifugation. After centrifugation, stratification can be observed. Use a pipette to suck the PBMC in the middle layer and the granulocytes distributed above the red blood cells at the bottom. To a 50mL centrifuge tube, add red blood cell lysate, place at room temperature for 10 minutes, centrifuge and discard the supernatant to obtain granulocytes. Wash with FACS buffer ((PBS+2%FBS) once, count and add ¹⁰⁶ cells/well to the 96-well U bottom plate. Add gradiently diluted antibodies to granulocytes and incubate at 4°C for 1 hour. Wash twice with FACS buffer, add secondary antibody (PE anti-human-IgG Fc antibody, Cat#409304, Bio Legend) and incubate at 4°C for half an hour, then perform flow detection on FACS CantoII to read the fluorescence signal value.

The results showed (see Figure 1), hu63-18T, hu67-18T, and hu67-19B, like the negative control antibody, did not bind to granulocytes under three different concentration conditions, while the control antibody A5B7-IgG1 (the antibody A5B7 The partial sequence of the antigen-binding part is derived from WO2007071422) which binds to granulocytes and exhibits a concentration-dependent effect. In the same experiment, anti-CEACAM1 (APC anti-CD66a antibody, Cat#10822-MM02-A, Sino Biological Inc), anti-CEACAM6 (CE ACAM6/CD66c antibody (PE), Cat#10823-R408-P, Sino Biological In) and anti-CEACAM8 (CE ACAM8/CD66b antibody (APC), Cat#11729-MM04-A, Sino Biological In) antibodies bound to granulocytes showed obvious positive signals, indicating that CEACAM1 is indeed on the surface of granulocytes , CEACAM6 and CEACAM8 expression.

Test Example 4. In vitro PBMC killing experiment

Bispecific antibody-mediated PBMC killing experiment on tumor cells was evaluated by quantitatively detecting the activity of apoptosis signal Caspase3/7.

Place the tumor cells in a ^{96-well plate with a white transparent bottom at a density of 3×10 5} cells/mL, 100 μL/well, and set aside blank control wells with medium and no cells, and place them at 37°C, 5% Incubate in a CO ₂ incubator for 20-24 hours. On the second day, 100 mL of fresh human peripheral blood was drawn, and PBMCs were separated by density gradient centrifugation according to the instructions of Ficoll Paque Premium 1.077. The PBMCs were resuspended in RPMI1640+10% FBS complete medium, and the density was adjusted to 1×10 after counting. ⁶ cells/mL. Take out the tumor cell plate from the previous day, take out 95 μL of medium per well, and then add 90 μL/well of PBMC cell suspension. Subsequently, 10 μL of antibody diluted in concentration was added to 90 μL of cell suspension. The control wells with antibody sample concentration of 0 were used as negative control wells, and the blank control wells with medium and no antibody were reserved, and 200nM RG7802 was added. The positive control wells were placed in a 37°C, 5% CO ₂ incubator for 48 hours. On the fourth day, take out the cell culture plate, place it at room temperature for 5-10 minutes, add 50μL of Caspase-Glo 3/7 reagent to each well with a Multidrop automatic dispenser, and read the chemiluminescence signal value with a microplate reader. Use Graphpad Prism 5 to process and analyze the data.

For tumor cells with a CEA binding site greater than 15000 (MKN45, HPAFII, LS174T and KatoIII), use the following formula to calculate the killing activity of each molecule, and define the 200nM RG7802 corresponding to a 96-well plate as 100%:

%200nM RG7802 cell lysis=(signal _{sample well} -signal _{negative control well} )/(signal _{200nM RG7802 —signal negative control well} ).

For tumor cells with CEA binding sites less than 15000 or CEA negative (HCC1954, HT29 and HCT116), the reading value of the negative control well is defined as 0%,

% Lysis = (signal _{sample well} -signal _{negative control well} )/signal _{negative control well} -signal _{blank control well} ).

The cell sources are as follows: MKN45 cells (Nanjing Kebai Biotechnology Co., Ltd., Cat#CBP60488), HPAFII (ATCC, CRL-1997), LS174T (ATCC, CL-188), KatoIII (ATCC, HTB-103), HCT116 (ATCC) ,CCL-247).

Killing activity on CEA positive tumor cells

A variety of CEA-positive and CEA-negative tumor cell lines were selected, and the cytotoxicity of PBMC mediated by bispecific antibodies on these tumor cells was tested. The results showed that hu67-18T, hu67-19B and hu63-18T all showed significant killing activity against CEA-positive tumor cell lines, but not against CEA-negative tumor cell lines. The negative control antibody (the sequence of the anti-CEA antibody in hu67-19B is replaced with the sequence of the anti-luciferase antibody) has no killing activity on all cell lines, indicating the performance of hu67-18T, hu67-19B and hu63-18T The killing activity of CEA is mediated by the CEA target.

Table 7. Killing activity of bispecific antibodies against different tumor cell lines

Test Example 5. In vitro T cell activation experiment-activation of Jurkat cell line

The bispecific antibody activates the Jurkat recombinant cell line and detects the expression of the NFAT-driven luciferase reporter gene (luciferase) after CD3 on the Jurkat cell surface is activated.

Inoculate tumor cell lines LS174T (CEA positive) and HCT116 (CEA positive) in a 96-well plate with a white transparent bottom, 3×10 ⁵ cells/mL, 100 μL/well, and place in a 37°C, 5% CO ₂ incubator for 20～ 24h. After removing the tumor cell culture supernatant the next day, add Jurkat-Lucia ^TM NFAT cell (invivogen, rep-qlcg5) cell suspension (6×10 ⁵ cells/mL, 75 μL/well), and serially diluted antibodies (25 μL/well). Well), and is equipped with a negative control well and a blank control well (only medium, no cells or antibodies) with an antibody concentration of 0. For the Jurkat cell activation experiment without tumor cells, the Jurkat cell suspension and serially diluted antibodies were directly added to the blank 96-well plate the next day. The cells were placed in a 37°C, 5% CO ₂ incubator and cultured for 5-6 hours. After the co-cultivation, take out the cell culture plate and place it at room temperature for 5-10 minutes, add 50μL Quanti-Luc Gold Detection Reagent (Invivogen, rep-qlcg5) to each well, and read the chemiluminescence signal with a multifunctional microplate reader value.

The results show that hu63-18T, hu67-19B and hu67-18T can effectively activate the Jurkat-Lucia ^TM NFAT cell line in the presence of CEA-positive cells LS174T, while the negative control antibody cannot activate the Jurkat-Lucia ^TM NFAT cell line (See Figure 2A). In the presence of the CEA-negative tumor cell line HCT116 or tumor-free cell line, none of the bispecific antibodies can activate the Jurkat-Lucia ^™ NFAT cell line, similar to the negative control antibody (see Figure 2B and Figure 2C). It shows that the activation of hu63-18T, hu67-19B and hu67-18T on Jurkat cells is CEA target-specific, and the degree of activation of non-CEA target-specific Jurkat cells is very low, indicating the good safety of bispecific antibody molecules. .

Test Example 6. Pharmacodynamic experiment of mouse LS174T model reconstructed by human PBMC

This test example uses the human PBMC reconstructed NDG mouse (Beijing Biocytogene Biotechnology Co., Ltd.) LS174T model (ATCC, CL-188) to evaluate the anti-CD3-CEA bispecific antibody tested in the present invention in mice. Tumor efficacy.

Mix LS174T cells (5×10 ⁶ cells/mouse/100μL) in vitro with freshly drawn PBMCs and inoculate them under the skin of the right ribs of NDG mice. When the tumor volume of tumor-bearing mice reaches about 80-100mm ³ , it will decrease The mice were randomly divided into groups, 7-8 mice in each group. The day of grouping was defined as Day 0 of the experiment, and the antibodies were injected intraperitoneally on the 1, 5, and 8 days. Monitor the tumor volume and animal weight twice a week and record the data.

The anti-tumor effect of hu63-18T (Figure 3) in vivo showed a dose-dependent effect. On the 15th day, the anti-tumor rate at 0.44mpk dose was 67.65% (p<0.001). The average tumor volume of the 2.19mpk dose group was smaller than that of the group. The volume and the tumor inhibition rate were greater than 100% (p<0.001), and the tumors in 6 out of 8 mice completely resolved. By the 21st day of observation, the tumors that had resolved in the 2.19mpk dose group still showed no signs of recurrence.

The anti-tumor effect of hu67-19B (Figure 3) in vivo showed a dose-dependent effect. On the 15th day, the anti-tumor rates at 0.1mpk and 0.5mpk doses were 48.45% (p<0.01) and 98.36% (p<0.001), respectively.

The anti-tumor effect of hu67-18T (Figure 3) in vivo showed a dose-dependent effect. On the 15th day, the anti-tumor rates at 0.44mpk and 2.19mpk were 68.34% (p<0.001) and 98.61% (p<0.001), respectively.

As a positive control, on the 15th day, the tumor inhibition rates of RG7802 at 0.5mpk and 2.5mpk doses were 11.12% and 59.38% (p<0.001).

Table 8. In vivo tumor inhibition rate of bispecific antibodies

Claims (17)

1. A bispecific antibody comprising an anti-CEA antibody portion and an anti-CD3 antibody portion, the anti-CEA antibody portion including the heavy chain variable region and the light chain variable region of an anti-CEA antibody, wherein:

   a) The amino acid sequences of HCDR1, HCDR2, and HCDR3 of the heavy chain variable region are shown in SEQ ID NOs: 7, 8, and 9, respectively, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 of the light chain variable region, respectively As shown in SEQ ID NO: 10, 11 and 12; or

   b) The amino acid sequences of HCDR1, HCDR2, and HCDR3 of the heavy chain variable region are shown in SEQ ID NOs: 13, 14 and 15, respectively, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 of the light chain variable region, respectively As shown in SEQ ID NO: 16, 17 and 18; or

   c) The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are shown in SEQ ID NOs: 7, 19 and 9, respectively, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region respectively As shown in SEQ ID NO: 10, 11, and 12.
2. The bispecific antibody according to claim 1, wherein the anti-CD3 antibody portion comprises a heavy chain variable region and a light chain variable region of an anti-CD3 antibody, wherein:

   The amino acid sequences of HCDR1, HCDR2, and HCDR3 of the heavy chain variable region are shown in SEQ ID NOs: 37, 38, and 39, respectively, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 of the light chain variable region are shown in SEQ ID NOs: 37, 38, and 39, respectively. ID NO: shown in 40, 41 and 42.
3. The bispecific antibody of claim 1 or 2, wherein the anti-CEA antibody portion comprises a heavy chain variable region and a light chain variable region as shown below, wherein:

   d) The amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO: 20, or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 20; and/or

   The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 21, or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 21; or

   e) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 22, or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 22; and/or

   The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 23, or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 23.
4. The bispecific antibody of any one of claims 1 to 3, wherein the anti-CD3 antibody portion comprises a heavy chain variable region and a light chain variable region of an anti-CD3 antibody, wherein:

   The heavy chain variable region of the anti-CD3 antibody has the same sequence as the heavy chain variable region contained in SEQ ID NO: 36, and/or

   The light chain variable region of the anti-CD3 antibody has the same sequence as the light chain variable region contained in SEQ ID NO: 36; preferably,

   The amino acid sequence of the heavy chain variable region of the anti-CD3 antibody is shown in SEQ ID NO: 52; and/or the amino acid sequence of the light chain variable region of the anti-CD3 antibody is shown in SEQ ID NO: 51.
5. The bispecific antibody according to any one of claims 1 to 4, wherein the anti-CEA antibody portion is an IgG type antibody, wherein,

   f) The antibody comprises two heavy chains with different amino acid sequences and two light chains with the same amino acid sequence, wherein the amino acid sequences of the first heavy chain and the second heavy chain are shown in SEQ ID NOs: 28 and 29, or It has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 28 and 29, respectively; and the amino acid sequence of the light chain is shown in SEQ ID NO: 31, or with the amino acid sequence shown in SEQ ID NO: 31 The sequence has at least 80% sequence identity; or

   g) The antibody comprises two heavy chains with the same amino acid sequence and two light chains with the same amino acid sequence, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO: 30, or is the same as that shown in SEQ ID NO: 30 The amino acid sequence has at least 80% sequence identity; and the light chain amino acid sequence is shown in SEQ ID NO: 31, or has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 31; or

   h) The antibody comprises two heavy chains with different amino acid sequences and two light chains with the same amino acid sequence, wherein the amino acid sequences of the first heavy chain and the second heavy chain are shown in SEQ ID NOs: 32 and 33, or It has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 32 and 33; and the light chain amino acid sequence is shown in SEQ ID NO: 35, or is similar to the amino acid sequence shown in SEQ ID NO: 35 Have at least 80% sequence identity; or

   i) The antibody comprises two heavy chains with the same amino acid sequence and two light chains with the same amino acid sequence, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO: 34 or is shown in SEQ ID NO: 34 The amino acid sequence has at least 80% sequence identity; and the light chain amino acid sequence is shown in SEQ ID NO: 35, or has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 35.
6. The bispecific antibody according to any one of claims 1 to 5, wherein the anti-CD3 antibody portion is an anti-CD3 single-chain antibody, and the amino acid sequence of the single-chain antibody is shown in SEQ ID NO: 36.
7. The bispecific antibody according to claim 6, wherein the heavy chain of the anti-CEA antibody and the anti-CD3 single-chain antibody are connected by a peptide bond or a linker.
8. The bispecific antibody according to any one of claims 1 to 7, wherein the bispecific antibody has an IgG-(scFv) 2 structure or an IgG-scFv structure.
9. The bispecific antibody according to claim 8, wherein the bispecific antibody having an IgG-(scFv)2 structure comprises chain 1 and chain 2,

   The chain 1 comprises VH(CEA)-CH1-Fc-scFv(CD3) from the amino terminus to the carboxyl terminus, wherein VH(CEA)-CH1-Fc is fused to scFv(CD3) directly or through a linker; and

   The chain 2 contains VL(CEA)-CL from the amino terminal to the carboxy terminal; preferably,

   j) The amino acid sequence of chain 1 is shown in SEQ ID NO: 44, or has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 44, and the amino acid sequence of chain 2 is shown in SEQ ID NO : Shown in 31, or at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 31; or

   k) The amino acid sequence of chain 1 is shown in SEQ ID NO: 46, or has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 46, and the amino acid sequence of chain 2 is shown in SEQ ID NO : 35, or at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 35.
10. The bispecific antibody according to claim 8, wherein the bispecific antibody having an IgG-scFv structure comprises chain 1, chain 2 and chain 3, wherein:

    The chain 1 contains VH(CEA)-CH1-Fc(knob)-scFv(CD3) from the amino terminus to the carboxyl terminus, wherein VH(CEA)-CH1-Fc(knob) is fused to scFv(CD3) directly or through a linker ；

    The chain 2 includes VL(CEA)-CL from the amino terminus to the carboxy terminus; and

    The chain 3 includes VH(CEA)-CH1-Fc(hole) from the amino terminus to the carboxy terminus; or

    The chain 1 contains VH(CEA)-CH1-Fc(hole)-scFv(CD3) from the amino terminus to the carboxyl terminus, wherein VH(CEA)-CH1-Fc(hole) is fused to scFv(CD3) directly or through a linker ；

    The chain 2 includes VL(CEA)-CL from the amino terminus to the carboxy terminus; and

    The chain 3 contains VH(CEA)-CH1-Fc(knob) from the amino terminus to the carboxy terminus; preferably,

    l) The amino acid sequence of chain 1 is shown in SEQ ID NO: 28, or has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 28, and the amino acid sequence of chain 3 is shown in SEQ ID NO: 43, or at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 43, and the amino acid sequence of chain 2 as shown in SEQ ID NO: 31, or shown in SEQ ID NO: 31 The amino acid sequence of has at least 80% sequence identity; or

    m) The amino acid sequence of chain 1 is shown in SEQ ID NO: 32, or has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 32, and the amino acid sequence of chain 3 is shown in SEQ ID NO: 45, or at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 45, and the amino acid sequence of chain 2 as shown in SEQ ID NO: 35, or shown in SEQ ID NO: 35 The amino acid sequence has at least 80% sequence identity.
11. An isolated nucleic acid molecule encoding the bispecific antibody of any one of claims 1-10.
12. A host cell comprising the nucleic acid molecule according to claim 11.
13. A pharmaceutical composition containing a therapeutically effective amount of the bispecific antibody according to any one of claims 1 to 10, or the nucleic acid molecule according to claim 11, and one or more pharmaceutically Acceptable carriers, diluents, buffers or excipients.
14. The use of the bispecific antibody according to any one of claims 1 to 10, or the nucleic acid molecule according to claim 11, or the pharmaceutical composition according to claim 13 in the preparation of a medicament for the treatment of cancer, wherein The cancer is preferably a CEA positive cancer.
15. The use according to claim 14, wherein the cancer is selected from melanoma, bowel cancer, non-small cell lung cancer (NSCLC), gastric cancer, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, cervical cancer, prostate cancer, kidney cancer , Bladder cancer, gallbladder cancer, esophageal cancer and medullary thyroid cancer.
16. A method of treating a disease associated with CEA-positive cells, the method comprising administering to a subject a therapeutically effective amount of the bispecific antibody of any one of claims 1 to 10, or the method of claim 11 Nucleic acid molecule, the pharmaceutical composition of claim 13, wherein the disease is preferably a CEA positive tumor or cancer.
17. The method for treating diseases associated with CEA-positive cells according to claim 16, wherein the CEA-positive cell-related diseases are selected from melanoma, bowel cancer, non-small cell lung cancer (NSCLC), gastric cancer, pancreatic cancer, breast cancer, and lung cancer , Ovarian cancer, cervical cancer, prostate cancer, kidney cancer, bladder cancer, gallbladder cancer, esophageal cancer and medullary thyroid cancer.

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