WO2023169583A1

WO2023169583A1 - Preparation and application of bispecific cell engager molecule constructed based on pep42

Info

Publication number: WO2023169583A1
Application number: PCT/CN2023/081116
Authority: WO
Inventors: 赵旭东; 曾筱茱
Original assignee: 四川大学华西医院
Priority date: 2022-03-11
Filing date: 2023-03-13
Publication date: 2023-09-14
Also published as: CN115304680A; CN115304680B

Abstract

The present invention provides a bispecific cell engager molecule. Specifically, the present invention provides a bispecific cell engager molecule comprising a first binding domain targeting a Pep42 receptor, a connecting segment, and a second binding domain of an anti-human CD3 molecule; connection to tumor cells is achieved by means of a ligand segment, and an anti-CD3 antibody segment connects to T lymphocytes, thus effectively joining effector immune cells with tumor cells, causing the T cells to target and kill the tumor cells; the invention has application potential in the preparation of anti-tumor drugs.

Description

Preparation and application of bispecific cell adapter molecules constructed based on Pep42

Technical field

The invention relates to the field of biomedicine. In particular, the present invention relates to the preparation of bispecific cell adapter molecules and their use.

Background technique

Cytotoxic T lymphocytes (CTL) play a key role in the immune response to cancer therapy. Because tumor cells can create an immunosuppressive environment through their own secretion of relevant cytokines or interaction with the tumor microenvironment, the CTL cells in the tumor microenvironment become dysfunctional. One strategy to modulate immune cells is to use bispecific cell engagers to activate immune cells and kill target cells, such as tumor cells. One type of bispecific cell engager is called Bispecific T-cell Engager (BiTE). It is named after its specific anti-antigen expressed by T cells at one end, which can guide T cells to kill the target. cells, this process is accompanied by the formation of transient cytolytic synapses between T cells and target tumor cells, followed by T cell proliferation and activation leading to tumor cell lysis.

Bispecific cell adapter molecules are composed of two protein or peptide sequences (antibodies being the most common) that bind different target proteins.

Glucose-regulated protein 78 (GRP78), also known as Bip protein, is encoded by the HSPA5 gene and is a key molecule in the unfolded protein response of the endoplasmic reticulum. Endoplasmic reticulum stress is a form of response of cells to the accumulation of proteins in the endoplasmic reticulum. It can induce the unfolded protein response (UPR), that is, cells reduce protein synthesis, promote protein degradation, and increase the expression of endoplasmic reticulum chaperones. Relieve endoplasmic reticulum stress. Endoplasmic reticulum stress that lasts too long or is too strong exceeds the ability of cells to regulate their own unfolded protein response, which will cause cellular metabolic disorders and apoptosis. In the tumor microenvironment, the presence of adverse factors such as hypoxia, glucose starvation, and acidosis often cause endoplasmic reticulum stress reactions. Tumor cells adapt to these adverse conditions and avoid death by activating the unfolded protein response. Under endoplasmic reticulum stress, GRP78 can be expressed in large amounts and promote the correct folding of proteins, relieve endoplasmic reticulum stress, and has a strong anti-apoptotic ability. It has been confirmed that GRP78 is up-regulated in various solid tumor cells such as lung cancer, liver cancer, and colorectal cancer, and is partially transferred to the cell membrane surface to participate in the activation and regulation of signaling pathways such as PI3K/AKT and JAK2/STAT3. The cell membrane transfer property of GRP78 is rarely seen in normal cells, suggesting that csGRP78 can be used as a tumor treatment target with good specificity and safety. However, there are currently no bispecific cell adapter molecules targeting GRP78.

Therefore, there is a need in the field to develop a bispecific cell adapter molecule targeting GRP78.

Contents of the invention

The purpose of the present invention is to provide a bispecific cell adapter molecule targeting GRP78.

In a first aspect of the invention, a cell engager molecule is provided, the cell engager molecule comprising:

(a) a first binding domain, the first binding domain specifically binds to the Pep42 receptor, and the first binding domain has a cyclic peptide structure; and

(b) A second binding domain that specifically binds CD3.

In another preferred embodiment, the cell adapter molecule is a bispecific cell adapter molecule.

In another preferred embodiment, the cyclic peptide structure is derived from the small molecule cyclic peptide Pep42.

In another preferred embodiment, the Pep42 receptor is Grp78.

In another preferred example, the first binding domain is derived from the Pep42 ligand peptide, and the Pep42 ligand peptide has the sequence shown in SEQ ID NO: 2 (CTVALPGGYVRVC).

In another preferred embodiment, the amino acid sequence of the first binding domain is shown in SEQ ID NO: 2.

In another preferred embodiment, the first binding domain forms a cyclic peptide structure through a disulfide bond between Cys at position 1 and Cys at position 13.

In another preferred embodiment, the second binding domain specifically binds human CD3.

In another preferred embodiment, the second binding domain has a peptide segment derived from an anti-human CD3 antibody.

In another preferred example, the second binding domain includes a VH segment, and the VH segment has the following complementarity determining region CDR:

VH-CDR1 shown in SEQ ID NO:5,

VH-CDR2 shown in SEQ ID NO:6, and

VH-CDR3 shown in SEQ ID NO:7; and/or

The second binding domain includes a VL segment, and the VL segment has the following complementarity determining region CDR:

VL-CDR1 shown in SEQ ID NO:8,

VL-CDR2 shown in SEQ ID NO:9, and

VL-CDR3 shown in SEQ ID NO:10;

Furthermore, any amino acid sequence in the above-mentioned CDR sequence also includes a derivative antibody composed of a heavy chain and a light chain, optionally adding, deleting, modifying and/or substituting at least one amino acid, so as to contain the derived CDR sequence. Derived sequences capable of retaining CD3 binding affinity.

In another preferred embodiment, the binding domain has the structure of a single domain antibody (sdAb), a single chain variable fragment (scFv), a Fab fragment, a ligand, a multimer thereof, or a combination thereof.

In another preferred embodiment, the VH segment has the amino acid sequence shown in SEQ ID NO:4, or has at least 80%, 85%, 90%, 91%, 92%, An amino acid sequence that has 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity; and/or

The VL segment has the amino acid sequence shown in SEQ ID NO: 9, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94% of the amino acid sequence shown in SEQ ID NO: 9 Amino acid sequences with %, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity.

In another preferred embodiment, the cell adapter molecule is a single-chain structure.

In another preferred embodiment, the cell adapter molecule has a structure selected from the following formula (I) or (II) from N-terminus to C-terminus:
S-D1-L1-D2-T (I); and
S-D2-L1-D1-T (II),

In the formula,

Each "-" is independently a connecting peptide or peptide bond;

S is none or signal peptide sequence;

D1 is the first binding domain;

L1 is none or the first connecting peptide;

D2 is the second binding domain;

T is None or Tagged protein.

In another preferred embodiment, the S is a signal peptide derived from mammalian Igκ.

In another preferred embodiment, the amino acid sequence of S is shown in SEQ ID NO: 18.

In another preferred example, the marker protein T is selected from: His tag, GGGS sequence, and FLAG tag.

In another preferred embodiment, the amino acid sequence of T is shown in SEQ ID NO: 17.

In another preferred embodiment, the amino acid sequence of L1 is shown in SEQ ID NO: 16.

In another preferred example, the amino acid sequence of D1 is as shown in SEQ ID NO:2; or the sequence identity with SEQ ID NO:2 is ≥85%, preferably ≥90%, more preferably ≥93% , or have a difference of 1, 2 or 3 amino acids compared with SEQ ID NO:2, and have the same or similar function as the sequence shown in SEQ ID NO:2.

In another preferred example, the D2 has a VH-L2-VL or VL-L2-VH structure from the N end to the C end, where VH is the VH segment, VL is the VL segment, and L2 is None or second linker peptide.

In another preferred embodiment, the amino acid sequence of L2 is shown in SEQ ID NO: 13.

In another preferred embodiment, the amino acid sequence of the bispecific cell adapter molecule is selected from:

(i) The amino acid sequence shown in SEQ ID NO: 20;

(ii) On the basis of the sequence shown in SEQ ID NO: 20, one or more amino acid residues are replaced, deleted, changed or inserted, or 1 to 30 amino acid residues are added to its N-terminal or C-terminal , preferably 1 to 10 amino acid residues, more preferably 1 to 5 amino acid residues, so that the amino acid residue obtained and the obtained amino acid sequence has ≥85% (preferably ≥90%, more preferably ≥95%, such as ≥96%, ≥97%, ≥98%) with the sequence shown in SEQ ID NO:20 or ≥99%) sequence identity; and the obtained amino acid sequence has the same or similar function as the sequence shown in (i).

In a second aspect of the present invention, a recombinant protein is provided, said recombinant protein comprising the cell adapter molecule as described in the first aspect of the present invention.

In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

In another preferred embodiment, the recombinant protein is a monomer, dimer, or multimer.

In another preferred embodiment, the recombinant protein is specific against Pep42 receptor and CD3.

In another preferred embodiment, the recombinant protein is a fusion protein.

In another preferred embodiment, the fusion protein is a bispecific antibody or a multispecific antibody (such as a trispecific antibody).

In another preferred embodiment, the multispecific antibody can not only bind to Pep42 receptor and CD3 simultaneously, but also specifically bind to additional target antigens (such as other tumor antigens, preferably other antigens of pancreatic cancer).

In a third aspect of the invention there is provided a polynucleotide encoding a polypeptide selected from the group consisting of:

(1) The cell adapter molecule according to the first aspect of the present invention; or

(2) The recombinant protein according to the second aspect of the present invention.

In another preferred embodiment, the sequence of the polynucleotide is shown in positions 1-894 of SEQ ID NO: 19.

In a fourth aspect of the present invention, a vector is provided, which vector contains the polynucleotide according to the third aspect of the present invention.

In another preferred embodiment, the vector includes: bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.

In another preferred example, the vector includes: pCDH, pTOMO, pGEM, pELNS, pMSGV, or a combination thereof.

In the fifth aspect of the present invention, an engineered host cell is provided, the host cell contains the vector as described in the fourth aspect of the present invention or the polynucleotide as described in the third aspect of the present invention is integrated into the genome.

In another preferred embodiment, the host cells are immune cells.

In another preferred embodiment, the host cells are in vivo cells and in vitro cultured cells that can be transplanted into cells in the body.

In another preferred embodiment, the cells cultured in vitro and transplantable into the body are selected from blood cells.

In another preferred embodiment, the immune cells are selected from the following group: T cells and NK cells.

In another preferred embodiment, the immune cells are from humans or non-human mammals (such as mice).

In a sixth aspect of the present invention, an antibody conjugate is provided, the antibody conjugate containing:

(a) an antibody portion selected from the group consisting of: a cell binder molecule according to the first aspect of the invention; and

(b) A coupling moiety coupled to the antibody moiety, the coupling moiety being selected from the group consisting of a detectable label, a drug, or a combination thereof.

In another preferred embodiment, the detectable label includes a radionuclide.

In another preferred embodiment, the drugs include toxins, cytokines, and enzymes.

In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computerized X-ray tomography) contrast agents, or can produce detectable Product enzymes, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, virus particles, liposomes, nanomagnetic particles, pro- Drug-activated enzymes (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (eg, cisplatin), or any form of nanoparticles, etc.

In another preferred embodiment, the antibody part and the coupling part are coupled through chemical bonds or linkers.

In another preferred embodiment, the immunoconjugate contains: a multivalent (such as bivalent) cell adapter molecule as described in the first aspect of the present invention.

In another preferred embodiment, the multivalent means that the amino acid sequence of the immunoconjugate contains multiple repeats of the cell adapter molecule as described in the first aspect of the present invention.

In the seventh aspect of the present invention, there is provided the use of an active ingredient selected from the following group: the cell adapter molecule as described in the first aspect of the present invention, the recombinant as described in the second aspect of the present invention Protein, the antibody conjugate according to the sixth aspect of the present invention, or a combination thereof, wherein the active ingredient is used to prepare a diagnostic reagent, a detection plate or a kit, and the reagent is used to detect Pep42 receptor and/or CD3.

In another preferred embodiment, the reagent, detection plate or kit is used to detect diseases related to abnormal expression or function of Pep42 receptor and/or CD3.

In another preferred embodiment, the reagent, detection panel or kit is used to predict the risk and/or prognosis of tumors.

In another preferred embodiment, the reagent is prepared as one or more reagents selected from the following group: isotope tracers, contrast agents, flow detection reagents, cell immunofluorescence detection reagents, nanomagnetic particles and display picture agent.

In another preferred embodiment, the reagent, detection plate or kit is used to screen drugs for treating GRP78-positive tumors.

In an eighth aspect of the present invention, a pharmaceutical composition is provided, which pharmaceutical composition contains:

(i) Active ingredient, the active ingredient is selected from the group consisting of: a cell adapter molecule as described in the first aspect of the present invention, a recombinant protein as described in the second aspect of the present invention, a cell adapter molecule as described in the fifth aspect of the present invention Host cells, antibody conjugates according to the sixth aspect of the present invention, or combinations thereof; and

(ii) One or more pharmaceutically acceptable carriers, diluents, fillers, binding agents, excipients, or combinations thereof.

In another preferred embodiment, the pharmaceutical composition is a liquid preparation.

In another preferred embodiment, the pharmaceutical composition is an injection.

In another preferred embodiment, the pharmaceutical composition includes 0.01 to 99.99% of the cell adapter molecule according to the first aspect of the present invention, the recombinant protein according to the second aspect of the present invention, the fifth aspect of the present invention. The host cell described in the aspect, the antibody conjugate as described in the sixth aspect of the present invention, or a combination thereof and 0.01 to 99.99% of the carrier, the percentage is the mass percentage of the pharmaceutical composition.

In another preferred embodiment, the pharmaceutical composition is used to prevent and/or treat diseases related to abnormal expression or function of Pep42 receptor and/or CD3.

In a ninth aspect of the present invention, a method for detecting Pep42 receptor and/or CD3 in a sample is provided, which method includes the steps:

(1) contacting the sample with the cell adapter molecule as described in the first aspect of the present invention;

(2) Detect whether the antigen-antibody complex is formed, where the formation of the complex indicates the presence of Pep42 receptor and/or CD3 in the sample.

In another preferred embodiment, the detection is for in vitro non-therapeutic and non-diagnostic purposes.

In a tenth aspect of the present invention, a composition for in vitro detection of Pep42 receptor and/or CD3 in a sample is provided, which includes the cell adapter molecule as described in the first aspect of the present invention, the cell adapter molecule as described in the second aspect of the present invention. The recombinant protein described above, the antibody conjugate as described in the sixth aspect of the present invention, the host cell as described in the fifth aspect of the present invention, or a combination thereof is used as an active ingredient.

In an eleventh aspect of the present invention, a detection plate is provided. The detection plate includes: a substrate (support plate) and a test strip. The test strip contains the cell junction as described in the first aspect of the invention. Organizer molecules, recombinant proteins as described in the second aspect of the present invention, antibody conjugates as described in the sixth aspect of the present invention, host cells as described in the fifth aspect of the present invention, or combinations thereof.

In a twelfth aspect of the present invention, a kit is provided, which includes:

(1) A first container containing the cell adapter molecule according to the first aspect of the present invention; and/or

(2) a second container containing a secondary antibody against the cell binder as described in the first aspect of the present invention;

Alternatively, the kit contains the detection plate according to the eleventh aspect of the present invention.

In a thirteenth aspect of the present invention, a method for preparing a recombinant polypeptide is provided, which method includes:

(a) Cultivate the host cell as described in the fifth aspect of the present invention under conditions suitable for expression;

(b) Isolating the recombinant polypeptide from the culture, the recombinant polypeptide being the cell adapter molecule according to the first aspect of the present invention or the recombinant protein according to the second aspect of the present invention.

In the fourteenth aspect of the present invention, there is provided a cell adapter molecule as described in the first aspect of the present invention, or a recombinant protein as described in the second aspect of the present invention, or an antibody conjugate as described in the sixth aspect of the present invention. , or the host cell according to the fifth aspect of the present invention, and/or the pharmaceutical composition according to the eighth aspect of the present invention, in the preparation of medicines for treating diseases related to abnormal expression or function of Pep42 receptor and/or CD3 the use of.

In another preferred embodiment, the abnormal expression of Pep42 receptor and/or CD3 refers to overexpression of Pep42 receptor and CD3.

In another preferred embodiment, the overexpression refers to the ratio of the expression level (F1) of Pep42 receptor and/or CD3 to the expression level (F0) under physiological conditions (ie, F1/F0) ≥ 1.5, preferably ≥ 2 , more preferably ≥2.5.

In another preferred embodiment, the drug is used to prevent and/or treat tumor occurrence, growth and/or metastasis.

In another preferred embodiment, the medicine is used to prevent and/or treat diseases.

In another preferred embodiment, the Pep42 receptor includes (but is not limited to) csGRP78.

In another preferred embodiment, the diseases associated with csGRP78 overexpression include: tumors, aging, cardiovascular disease, obesity, or combinations thereof.

In another preferred embodiment, the disease is a malignant tumor in which csGRP78 is overexpressed (ie, csGRP78 positive).

In another preferred embodiment, the tumors include hematological tumors and solid tumors.

In another preferred embodiment, the blood tumor is selected from the following group: acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), diffuse leukemia B-cell lymphoma (DLBCL), or combinations thereof.

In another preferred embodiment, the solid tumor is selected from the following group: breast cancer, gastric cancer, hepatobiliary cancer, colorectal cancer Bowel cancer, bladder cancer, non-small cell lung cancer, ovarian and esophageal cancer, glioblastoma, lung cancer, pancreatic cancer, prostate cancer, etc., or combinations thereof.

In another preferred example, the drug is used to inhibit GRP78-positive cells, preferably including: human pancreatic cancer cell line ASPC1, human pancreatic cancer cell line BXPC3, human pancreatic cancer cell line MHCC-97H, acute myeloid leukemia Cell line U937, acute myeloid leukemia cell line KG-1A, or combinations thereof.

In a fifteenth aspect of the present invention, a method for treating diseases related to abnormal expression or function of Pep42 receptor and CD3 is provided, by administering an effective amount of the cell engagement as described in the first aspect of the present invention to a subject in need. Organizer molecules, or recombinant proteins as described in the second aspect of the present invention, or host cells as described in the fifth aspect of the present invention, or antibody conjugates as described in the sixth aspect of the present invention, or as described in the eighth aspect of the present invention The pharmaceutical composition described in the aspect, or a combination thereof.

In another preferred embodiment, the diseases related to abnormal expression or function of Pep42 receptor include: tumors, aging, cardiovascular disease, obesity, or combinations thereof.

In another preferred embodiment, the disease associated with abnormal expression or function of the Pep42 receptor includes tumors, preferably including pancreatic cancer.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described one by one here.

Description of drawings

The following drawings are used to illustrate specific embodiments of the invention and are not intended to limit the scope of the invention as defined by the claims.

Figure 1 shows a schematic diagram of the VH and VL chains of OKT3 described in Journal of Biochemistry, 1996, 120: 657-662. Among them, positions 1-19 of the VH amino acid sequence are signal peptides, and positions 1-22 of the VL amino acid sequence are signal peptides.

Figure 2 shows a schematic diagram of the construction, expression and purification of GRP78-CD3/BiTE protein.

Figure 3 shows a schematic diagram of the binding ability of GRP78-CD3/BiTE to GRP78-positive cells or CD3-expressing T cells.

Figure 4 shows a schematic diagram of the killing ability of T cells against target cells mediated by GRP78-CD3/BiTE.

Figure 5 shows a schematic diagram of the ability of T cells to secrete cytokines mediated by GRP78-CD3/BiTE.

Figure 6 shows a schematic diagram of T cell phenotype proportions in each group.

Figure 7 shows a schematic diagram of the efficacy of bispecific cell engager molecules in mice.

Figure 8 shows a schematic diagram of the killing detection of clinical samples by bispecific cell adapter molecules.

Detailed ways

After extensive and in-depth research and extensive screening, the inventor developed for the first time the preparation and application of a bispecific cell adapter molecule constructed based on Pep42. Experimental results show that the BiTE targeting the GRP78 receptor of the present invention has a significant killing effect on target cells and a specific anti-tumor cell effect. On this basis, the present invention was completed.

The bispecific cell adapter molecule provided by the present invention consists of three parts: a first binding domain (ligand segment) targeting tumor cell surface antigen receptors, a connecting segment and a second binding domain of an anti-human CD3 molecule (antibody segment), connects to tumor cells through the ligand segment, and at the same time, the anti-CD3 antibody segment connects to T lymphocytes, thereby effectively attracting effector immune cells to the local tumor, allowing the body to exert anti-tumor effects more effectively.

the term

In order to make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise defined herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Before the present invention is described, it is to be understood that this invention is not limited to the specific methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, and that the scope of the invention will be limited only by the appended claims.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values between 99 and 101 and between (eg, 99.1, 99.2, 99.3, 99.4, etc.).

The three-letter codes and single-letter codes for amino acids used in the present invention are as described in J. biol. chem, 243, p3558 (1968).

As used herein, the term "treatment" refers to the administration of an internal or external therapeutic agent, including the bispecific cell engager molecules and compositions thereof of the present invention, to a patient having one or more symptoms of a disease for which it is known that the Therapeutic agents have a therapeutic effect on these symptoms. Typically, a therapeutic agent is administered to a patient in an amount effective to alleviate one or more symptoms of the disease (a therapeutically effective amount).

As used herein, the terms "optionally" or "optionally" mean that the subsequently described event or circumstance may occur but does not necessarily occur. For example, "optionally including 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a specific sequence may be present but are not required to be present, and may be 1, 2 or 3.

"Sequence identity" as used herein means the degree of identity between two nucleic acids or two amino acid sequences when optimally aligned and compared with appropriate substitutions, insertions, deletions and other mutations. The sequence identity between the sequence described in the present invention and its identical sequence may be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ,100%.

Bispecific cell adapter molecules

As used herein, the terms "bispecific cell engager molecule", "bispecific cell engager", "cell engager", "BiTE", "bispecific antibody", "antibody of the invention" are interchangeable Use, all means that the first aspect of the present invention provides a cell adapter molecule capable of binding Pep42 receptor and CD3 simultaneously.

Bispecific cell adapter molecules are composed of two protein or peptide sequences (antibodies being the most common) that bind different target proteins. The function of the BiTE of the present invention is determined by the gene-specific gene sequences of its Pep42 ligand segment and CD3 antibody segment. The antibody of the present invention can combine with Pep42 receptor and CD3 at the same time, connect to tumor cells through the ligand segment, and at the same time, the anti-CD3 antibody segment connects to T lymphocytes, thereby effectively connecting effector immune cells with tumor cells and exerting anti-tumor effects more effectively. . Using the VL, VH segment genes or complementarity determining region (CDR) genes of the present invention, different forms of genetically engineered antibodies can be transformed and produced in any expression system using prokaryotic and eukaryotic cells.

As used herein, the term "bispecific" refers to a molecule that contains at least two binding domains with different binding specificities. Each binding domain is capable of specifically binding to a target molecule. In some embodiments, a bispecific cell engager is a polymer molecule with two or more peptides. In some embodiments, the binding domain comprises the antigen-binding domain, or variable region, or CDR of an antibody. In some embodiments, the binding domain comprises a ligand or fragment thereof that specifically binds to the target protein.

At least two targeting domains of the cell adapter molecules of the invention are optionally linked by a linker peptide. The preferred connecting peptide sequence is shown in SEQ ID NO: 16, but is not limited to this.

GRP78 and endoplasmic reticulum stress response

Solid tumors have a unique tumor microenvironment. Due to poor vasculature, they are usually in a state of hypoxia and glucose starvation, and produce large amounts of lactic acid through the glycolysis pathway. In response to pressure stimuli in the tumor microenvironment, solid tumor cells cause endoplasmic reticulum stress, induce a self-protective response to unfolded proteins, reduce the secretion and accumulation of misfolded proteins, maintain endoplasmic reticulum homeostasis, and create survival opportunities for tumor cells. Chance. As a key regulatory protein of the unfolded protein response, GRP78 has a significantly increased expression level in tumor cells. Under normal circumstances, GRP78 binds to the luminal domain of the endoplasmic reticulum of IRE1, PERK and ATF6 proteins, inhibiting their functions. When the endoplasmic reticulum is stressed, GRP78 dissociates from the three proteins and binds to unfolded or misfolded proteins. On the one hand, misfolded proteins are transported back to the cytoplasm and degraded by 26S ubiquitinase. On the other hand, the energy of ATP hydrolysis is used to accelerate the folding of proteins, so that the correctly folded proteins are transported to the Golgi apparatus, thereby promoting the correct folding and synthesis of nascent proteins. Prevents the accumulation of misfolded, unfolded proteins. After dissociation, IRE1, PERK and ATF6 cause downstream unfolded protein responses through their own different signaling pathways.

GRP78, which is upregulated in tumor cells, can partially escape to the surface of tumor cell membranes and participate in processes such as tumor cell proliferation, invasion, migration, and drug resistance. Further studies found that the expression abundance of csGRP78 was related to related to the malignancy of tumor cells. Although the mechanism of GRP78 membrane transfer is not yet fully understood, and different cells may have different transfer mechanisms, this does not affect its application value in bispecific cell adapter therapy for solid tumors. In addition, csGRP78 expression can also be detected in blood tumor cells, such as U937 and KG-1A. Therefore, the bispecific cell adapter targeting csGRP78 of the present invention has important value in the treatment of solid tumors, hematological tumors or other diseases related to abnormal expression of csGRP78.

Pep42

The cyclic peptide form of Pep42 specifically binds to the csGRP78 receptor. In 2006, Kim et al. screened the csGRP78-specific small molecule cyclic peptide ligand Pep42 by using phage cyclic peptide library technology. The Pep42 cyclic peptide consists of 13 amino acids and has the sequence CTVALPGGYVRVC. Pep42 forms a ring through the formation of a disulfide bond between two cysteines at both ends. Mutation experiments further confirmed that the ring structure of Pep42 is the molecular basis for its specific recognition of csGRP78. Cyclic peptides are an important class of active peptides that exist in plants, animals and humans. They have a long half-life, a clear fixed conformation, and can bind well to receptors. In the human body, linear peptide ring formation mostly occurs between two cysteines, and the ring formation efficiency is high, which provides important conditions for the application of cyclic peptide drugs. At present, a large number of cyclic peptide ligands have been obtained based on gene encoding technology of artificial design and in vitro evolution, and Pep42 is one of them.

After Pep42 specifically binds to csGRP78, it can be internalized into cells and has no cytotoxicity. This provides a powerful tool for the design of drugs targeting csGRP78 to treat tumors, and has been proven to effectively deliver chemotherapy drugs and reduce the impact of chemotherapy drugs on normal cells. toxic effects.

Antibody

As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric protein of approximately 150,000 daltons with the same structural characteristics, consisting of two identical light chains (L) and two identical heavy chains (H) Composition. Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain (VH) at one end, followed by multiple constant domains. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. . Special amino acid residues form the interface between the variable regions of the light and heavy chains.

As used herein, the term "variable" means that certain portions of the variable regions of an antibody differ in sequence and contribute to the binding and specificity of each particular antibody to its particular antigen. However, variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments in the variable regions of the light and heavy chains called complementarity determining regions (CDRs) or hypervariable regions. The more conserved part of the variable region is called the framework region (FR). The variable regions of natural heavy and light chains each contain four FR regions, which are generally in a β-sheet configuration and are connected by three CDRs forming a connecting loop. In some cases, a partial β-sheet structure can be formed. The CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen of the antibody Binding site (see Kabat et al., NIH Publ. No. 91-3242, Vol. 1, pp. 647-669 (1991)). Constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as involvement in antibody-dependent cytotoxicity of the antibody.

The term "antibody fragment" or "antigen-binding fragment" is used to refer to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, single chain Fvs (scFv), single chain antibody, Disulfide-linked Fvs (sdFv), fragments containing VL or VH domains, fragments generated from Fab expression libraries, and anti-idiotypic (anti-Id) antibodies. Regardless of their structure, antibody fragments bind to the same antigen recognized by the intact antibody. The term "antibody fragment" includes DARTs and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein containing an immunoglobulin variable region that acts like an antibody by binding to a specific antigen to form a complex. "Single chain fragment variable region" or "scFv" refers to a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin. In some aspects, the region domain is linked to a short linker peptide of 10 to about 25 amino acids. The linker can be rich in glycine for flexibility and serine or threonine for solubility, and can connect the N-terminus of VH or the C-terminus of VL, or vice versa. Despite the removal of the constant region and the introduction of linkers, the protein retains the specificity of the original immunoglobulin. Regarding IgG, a standard immunoglobulin molecule contains two identical light chain polypeptides with a molecular weight of approximately 23,000 daltons and two identical heavy chain polypeptides with a molecular weight of 53,000-70,000. The four chains are usually linked by disulfide bonds in a "Y" configuration, with the light chain bracketing the heavy chain from the mouth of the "Y" and extending through the variable region.

As mentioned above, variable regions allow an antibody to selectively recognize and specifically bind to an epitope on an antigen. That is, the VL domain of an antibody and the VH domain or a subset of complementarity determining regions (CDRs) of the antibody combine to form a variable region that defines a three-dimensional antigen binding site. This quaternary antibody structure forms the antigen binding site present at the end of each arm of each Y configuration. More specifically, the antigen binding site is defined by three CDRs on each of the VH and VL chains (ie, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3). In some cases, for example, certain immunoglobulin molecules are derived from camelid species or engineered based on camelid immunoglobulins. Alternatively, an immunoglobulin molecule may be composed of only a heavy chain without a light chain or only a light chain without a heavy chain.

In naturally occurring antibodies, the six CDRs present in each antigen-binding domain are short, non-contiguous amino acid sequences that are specifically positioned to Forming an "antigen binding domain". The remaining amino acids in the antigen-binding domain, termed the "framework" domain, show less inter-molecular variability. The framework regions predominantly adopt a β-sheet conformation, and the CDRs form loops that connect and in some cases form part of the β-sheet structure. Therefore, the framework region serves to form a scaffold that positions the CDRs in the correct orientation through non-covalent interactions between chains. The antigen-binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes non-covalent binding of the antibody to its cognate epitope. Having been precisely defined, one of ordinary skill in the art can readily identify the CDRs and constructs for any given heavy or light chain variable region, respectively. amino acids in the shelf region.

As used herein, a "variant" of an antibody, antibody fragment, or antibody domain means an antibody, antibody fragment, or antibody domain that: (1) is at least 80%, 85% identical to the original antibody, antibody fragment, or antibody domain , 90%, 95%, 96%, 97%, 98% or 99% sequence identity, and (2) specifically binds to the same target as the original antibody, antibody fragment or antibody domain. It will be understood that where sequence identity is expressed in the form of "at least x% identical" or "at least x% identical," such embodiments include any and all numerical percentages at or above the lower limit. Furthermore, it should be understood that where an amino acid sequence is present in this application, it shall be construed as otherwise disclosed or included as being at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, identical to that amino acid sequence. At least 97%, at least 98% or at least 99% identical.

Included within the scope of the multispecific molecules of the invention are various compositions and methods including: asymmetric IgG-like antibodies (e.g., triomab/quadroma) ; buttonhole antibodies (knobs-into-holes antibodies); cross monoclonal antibodies (Cross MAb); electrostatically matched antibodies; LUZ-Y; chain exchange engineered domain (SEED) bodies; Fab exchange antibodies, symmetrical IgG antibodies ; Two-in-one antibody; cross-linked monoclonal antibody, mAb2; Cov X-body; dual variable domain (DVD)-Ig fusion protein; IgG-like bispecific antibody; Ts2Ab; BsAb; scFv/Fc fusion; Bi(scFv)2-Fabs; F(ab)2 fusion proteins; dual-acting or Bis-Fab; Dock-and-Lock (DNL); Fab-Fv; scFv-based antibodies and diabody-based antibodies (e.g., bispecific T cell engagers (BiTEs); tandem diabodies (Tandab); DARTs; single chain diabodies; TCR-like antibodies; human serum albumin scFv fusion proteins, COMBODIES and IgG/non-IgG fusion proteins.

The present invention includes not only complete monoclonal antibodies, but also antibody fragments with immunological activity, such as Fab or (Fab')2 fragments; antibody heavy chains; and antibody light chains.

The present invention is preferably in the form of a single chain antibody (scFv), a minimal antibody fragment containing an antibody heavy chain variable region, a light chain variable region, but no constant region, and having all antigen binding sites. Typically, Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding.

The term "epitope" or "antigenic determinant" refers to the site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes usually include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-consecutive amino acids in a unique spatial conformation. An epitope may be a discontinuous three-dimensional site on the antigen recognized by the antibody or antigen-binding fragment of the present invention.

The terms "specifically binds", "selectively binds", "selectively binds" and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen. Typically, antibodies bind with an affinity (KD) of about less than 10 ^"7 M, such as about less than 10 ^"8 M, 10 ^"9 M, or 10 ^"10 M or less.

The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of said antibodies. Similar things.

In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be obtained by standard recombinant DNA techniques and are useful antibodies. A chimeric antibody is a molecule in which the different parts are derived from different animal species, such as a chimeric antibody having a variable region from a mouse monoclonal antibody, and a constant region from a human immunoglobulin (see, e.g., U.S. Patent 4,816,567 and U.S. Patent 4,816,397, which is incorporated herein by reference in its entirety). Humanized antibodies refer to antibody molecules derived from non-human species, having one or more complementarity determining regions (CDRs) derived from non-human species and framework regions derived from human immunoglobulin molecules (see U.S. Patent 5,585,089, This article is incorporated by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using recombinant DNA techniques well known in the art.

In the present invention, antibodies may be monospecific, bispecific, trispecific, or more multispecific.

As used herein, the terms "heavy chain variable region" and "VH" are used interchangeably.

As used herein, the terms "variable region" and "complementarity determining region (CDR)" are used interchangeably.

The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contribute to antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat E.A et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242).

In a preferred embodiment of the present invention, the light chain of the antibody includes the above-mentioned light chain variable region and light chain constant region, and the light chain constant region can be of murine or human origin.

In the present invention, the antibody of the present invention also includes conservative variants thereof, which means that compared with the amino acid sequence of the antibody of the present invention, there are at most 10, preferably at most 8, more preferably at most 5, optimally Up to three amino acids are replaced by amino acids with similar or similar properties to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table 1.

Table 1

Furthermore, the amino acid sequence also includes a sequence formed by adding, deleting, modifying and/or substituting at least one amino acid sequence, preferably having a homology or sequence identity of at least 80%, preferably at least 85%, and more Preferably it is at least 90%, most preferably at least 95% of the amino acid sequence.

Methods for determining sequence homology or identity known to those of ordinary skill in the art include, but are not limited to: Computational Molecular Biology, edited by Lesk, A.M., Oxford University Press, New York, 1988; Biocomputing: Information Biocomputing: Informatics and Genome Projects, edited by Smith, D.W., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, edited by Griffin, A.M. and Griffin, H.G. , Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux , J. M Stockton Press, New York, 1991 and Carillo, H. and Lipman, D., SIAM J. Applied Math., 48:1073 (1988). The preferred method of determining identity is to obtain the greatest match between the sequences tested. Methods for determining identity are compiled in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to, the GCG package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S, F. et al., 1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, MD. 20894; Altschul, S. et al., 1990). The well-known Smith Waterman algorithm can also be used to determine identity.

Preferably, the antibody described herein is an antibody full-length protein, an antigen-antibody binding domain protein fragment, a bispecific antibody, a multispecific antibody, a single chain antibody fragment (scFv), a single domain antibody (single domain antibody) , sdAb) and one or more of single-domain antibodies (Signle-domain antibodies), as well as monoclonal antibodies or polyclonal antibodies prepared from the above antibodies. The monoclonal antibodies can be developed through a variety of approaches and technologies, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc. The mainstream method is to prepare monoclonal antibodies from wild-type or transgenic mice through hybridoma technology.

The full-length antibody protein is a conventional antibody full-length protein in the art, which includes a heavy chain variable region, a light chain variable region, a heavy chain constant region and a light chain constant region. The heavy chain variable region and light chain variable region of the protein together with the human heavy chain constant region and the human light chain constant region constitute a fully human antibody full-length protein. Preferably, the full-length antibody protein is IgG1, IgG2, IgG3 or IgG4.

The antibody of the present invention can be a double-chain or single-chain antibody, and can be selected from animal-derived antibodies, chimeric Antibodies, humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, more preferably fully humanized antibodies.

The antibody derivatives of the present invention can be single-chain antibodies and/or antibody fragments, such as: Fab, Fab', (Fab')2 or other known antibody derivatives in the field, as well as IgA, IgD, IgE , IgG and IgM antibodies or any one or more of other subtypes of antibodies.

In a preferred embodiment of the present invention, the bispecific cell adapter molecule is a single-chain antibody, which includes an anti-CD3 single-chain antibody segment, a connecting peptide and a Pep42 ligand segment, wherein the anti-CD3 single-chain antibody is one of the best in the field. Conventional single-chain antibodies include heavy chain variable regions and light chain variable regions.

In the present invention, the animal is preferably a mammal, such as a mouse.

The antibodies of the invention may be chimeric antibodies, humanized antibodies, CDR-grafted and/or modified antibodies targeting the Pep42 receptor and CD3 (eg, human Pep42 receptor and CD3).

In the above content of the present invention, the number of added, deleted, modified and/or substituted amino acids is preferably no more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably no more than 35%, and more preferably 1-33%. , more preferably 5-30%, more preferably 10-25%, more preferably 15-20%.

In the above content of the present invention, more preferably, the number of added, deleted, modified and/or substituted amino acids can be 1-7, more preferably 1-5, more preferably 1-3, even more preferably For 1-2 pieces.

Recombinant protein

The invention also provides a recombinant protein, which includes the antibody of the invention.

The recombinant protein is a conventional protein in this field. Preferably, it is an antibody full-length protein, an antigen-antibody binding domain protein fragment, a bispecific antibody, a multispecific antibody, a single chain antibody fragment (scFv). ), one or more of single domain antibody (single domain antibody, sdAb) and single region antibody (Signle-domain antibody), as well as monoclonal antibodies or polyclonal antibodies prepared from the above antibodies. The monoclonal antibodies can be developed through a variety of approaches and technologies, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc. The mainstream method is to prepare monoclonal antibodies from wild-type or transgenic mice through hybridoma technology.

The single-chain antibody is a conventional single-chain antibody in this field, which includes a heavy chain variable region, a light chain variable region, and a short peptide of 15 to 20 amino acids.

The antigen-antibody binding domain protein fragment is a conventional antigen-antibody binding domain protein fragment in the art, and includes the light chain variable region, the light chain constant region and the Fd segment of the heavy chain constant region. Preferably, the antigen-antibody binding domain protein fragments are Fab and F(ab').

Wherein, the preparation method of the recombinant protein is a conventional preparation method in this field. The preparation method is preferably: isolated from expression transformants that recombinantly express the protein or obtained by artificially synthesizing protein sequences. The preferred method for isolating and obtaining the expression transformant recombinantly expressing the protein is as follows: cloning the nucleic acid molecule encoding the protein and carrying a point mutation into a recombinant vector, and transforming the obtained recombinant vector into the transformant to obtain recombinant expression By culturing the recombinant expression transformant, the recombinant protein can be obtained by isolation and purification.

nucleic acid

The present invention also provides polynucleotide molecules encoding the above-mentioned cell adapter molecules. The polynucleotides of the invention may be in DNA form or RNA form. Forms of DNA include cDNA, genomic DNA, or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide may be identical to the coding region sequence of the antibody of the present invention or may be a degenerate variant. As used herein, "degenerate variant" in the present invention refers to a nucleic acid sequence encoding a nucleic acid sequence having the same amino acid sequence as the polypeptide of the present invention, but having a different sequence in its coding region.

Polynucleotides encoding mature polypeptides of the present invention include: coding sequences encoding only mature polypeptides; coding sequences for mature polypeptides and various additional coding sequences; coding sequences for mature polypeptides (and optional additional coding sequences) and non-coding sequences .

The term "polynucleotide encoding a polypeptide" may include polynucleotides encoding such polypeptides, or may also include polynucleotides that also include additional coding and/or non-coding sequences.

The invention also relates to polynucleotides that hybridize to the sequences described above and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The invention particularly relates to polynucleotides that hybridize under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) adding There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90%, more It is best when hybridization occurs only when the ratio is above 95%. Moreover, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 20.

The full-length nucleotide sequence of the cell adapter molecule of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. A feasible method is to use artificial synthesis to synthesize the relevant sequences, especially when the fragment length is short. Often, fragments with long sequences are obtained by first synthesizing multiple small fragments and then ligating them. In addition, its coding sequence and expression tag (such as 6His) can also be fused together to form a fusion protein.

carrier

The invention also provides a recombinant expression vector containing the nucleic acid.

The recombinant expression vector can be obtained by conventional methods in the art, that is, it is constructed by connecting the nucleic acid molecule of the present invention to various expression vectors. The expression vector is a variety of conventional vectors in the art, as long as it can accommodate the aforementioned nucleic acid molecules. The vector preferably includes: various plasmids, cosmids, phage or viral vectors, etc.

The present invention also provides a recombinant expression transformant comprising the above recombinant expression vector.

Wherein, the preparation method of the recombinant expression transformant is a conventional preparation method in this field, preferably: it is prepared by transforming the above recombinant expression vector into a host cell. The host cells are various conventional host cells in the art, as long as the recombinant expression vector can stably replicate itself and the nucleic acid carried can be effectively expressed. Preferably, the host cell is E.coli TG1 or E.coli BL21 cells (expressing single chain antibodies or Fab antibodies), or HEK293 or CHO cells (expressing full-length IgG antibodies). The preferred recombinant expression transformant of the present invention can be obtained by transforming the aforementioned recombinant expression plasmid into a host cell. The transformation method is a conventional transformation method in this field, preferably a chemical transformation method, a heat shock method or an electroporation method.

Preparation of cell adapter molecules

The method for preparing the sequence of the DNA molecule of the cell adapter molecule or its fragment of the present invention is preferably to fuse the coding sequences of the ligand segment and the antibody segment together to form a single-chain antibody. In addition, it can be obtained using conventional techniques, such as PCR amplification or genomic library screening.

Once the relevant sequence is obtained, recombination can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, transforming it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

In addition, artificial synthesis methods can also be used to synthesize relevant sequences, especially when the fragment length is short. Often, fragments with long sequences are obtained by first synthesizing multiple small fragments and then ligating them.

At present, the DNA sequence encoding the cell adapter of the present invention (or its fragment, or its derivative) can be obtained entirely through chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the invention through chemical synthesis.

The invention also relates to vectors comprising the appropriate DNA sequences as described above and appropriate promoter or control sequences. These vectors can be used to transform appropriate host cells to enable expression of the protein.

The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Preferred cells include (but are not limited to): T cells.

Typically, the transformed host cells are cultured under conditions suitable for expression of the antibodies of the invention. Then use conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrolysis The antibody of the present invention can be purified by conventional separation and purification means well known to those skilled in the art such as electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography.

The resulting cell adapters can be characterized by conventional means. For example, its binding specificity can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity can be determined, for example, by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

The cell adapter of the present invention can be expressed within the cell, on the cell membrane, or secreted outside the cell. If desired, the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, infiltration sterilization, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

Conventional preparation methods for bispecific cell adapter molecules in this field are as follows:

1. First use PCR to prepare the bispecific antibody gene, then clone this gene into the expression vector pRB199 and transform it into E. coli strain BL21 (λDE3) to prepare inclusion bodies. Subsequently, 6M guanidine-HCl and DET (dithioerythritol) were added to the inclusion body for denaturation, and then diluted 100% with the renaturation buffer, mixed quickly at 4°C, and then incubated at 4°C for 72 hours to refold the protein. After renaturation, 0.1M Tris and 0.5M NaCl were added at a ratio of 1:10 for dialysis, repeated three times and then filtered (0.2μm), followed by metal ion affinity chromatography. Subsequently, a fast protein liquid chromatograph (BioLogic DuoFlow 10System; Bio-Rad) was used for purification, and a Histidine tag fusion protein purification column was used for separation. Proteins were eluted by a stepwise gradient of imidazole at a flow rate of 1 ml/min. The product was processed through a column (Sartorius Stedim Biotech) to remove proteins with a molecular weight greater than 10,000, dialyzed against PBS, and filtered to sterilize. After measuring the concentration, use SDS/PAGE for silver staining identification (see PNAS, 2013, 110(1):270-275);

2. Infect CHO cells with lentivirus containing bispecific antibodies and culture them for 72 hours after infection. Fluorescent expression of CHO cells can be observed. The successfully infected cell lines were expanded and cultured. CHO cells stably expressing bispecific antibodies can continue to secrete and express them. Cell supernatants were collected for protein purification and concentration. Subsequently, a fast protein liquid chromatograph (BioLogic DuoFlow 10System; Bio-Rad) was used for purification, and a Histidine tag fusion protein purification column was used for separation. Pass five times the volume of the sample's equilibration buffer through the nickel column at a flow rate of 0.5-1ml/min. After equilibration, the sample was flowed through the nickel column at a flow rate of 0.5 ml/min. Wash the nickel column with five times the volume of equilibration buffer to remove background protein until the absorbance of the eluent at 280 nm is 0. The target protein is eluted by imidazole at a flow rate of 0.5ml/min. Then use an ultrafiltration tube to concentrate the protein and replace the salt solution. After measuring the concentration, use western blot for identification (see Oncoimmunology, 2015, 4(4):e989776.).

Those skilled in the art can make routine selections or equivalent modifications to the above methods to prepare or produce Bispecific cell engager molecules of the invention.

Antibody-drug conjugates (ADCs)

The invention also provides antibody-drug conjugates (ADCs) based on the antibodies of the invention.

Typically, the antibody-conjugated drug includes the antibody and an effector molecule, and the antibody is coupled to the effector molecule, and preferably is chemically coupled. Among them, the effector molecule is preferably a drug with therapeutic activity. In addition, the effector molecule may be one or more of toxic proteins, chemotherapeutic drugs, small molecule drugs or radionuclides.

The antibody of the present invention and the effector molecule can be coupled through a coupling agent. Examples of the coupling agent may be any one or more of non-selective coupling agents, coupling agents utilizing carboxyl groups, peptide chains, and coupling agents utilizing disulfide bonds. The non-selective coupling agent refers to a compound that allows the effector molecule and the antibody to form a covalent bond, such as glutaraldehyde, etc. The coupling agent utilizing carboxyl groups may be any one or more of aconitic anhydride coupling agents (such as aconitic anhydride) and acyl hydrazone coupling agents (the coupling site is an acyl hydrazone).

Certain residues on antibodies (such as Cys or Lys, etc.) are used to connect to a variety of functional groups, including imaging reagents (such as chromophores and fluorescent groups), diagnostic reagents (such as MRI contrast agents and radioisotopes) , stabilizers (e.g. glycol polymers) and therapeutic agents. Antibodies can be coupled to functional agents to form antibody-functional agent conjugates. Functional agents (eg drugs, detection reagents, stabilizers) are coupled (covalently linked) to the antibody. The functional agent can be linked to the antibody directly or indirectly through a linker.

Antibodies can be conjugated with drugs to form antibody drug conjugates (ADCs). Typically, ADCs contain a linker between the drug and the antibody. Linkers can be degradable or non-degradable linkers. Degradable linkers are typically susceptible to degradation in the intracellular environment, such as at the target site, allowing the drug to be released from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers, including peptidyl-containing linkers that can be degraded by intracellular proteases, such as lysosomal or endosomal proteases, or sugar linkers, such as those that can be degraded by glucuronides. Enzymatic degradation of glucuronide-containing linkers. Peptidyl linkers may include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (eg, linkers that hydrolyze at pH less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (eg, disulfide linkers). Nondegradable linkers typically release the drug under conditions in which the antibody is hydrolyzed by proteases.

Before being connected to the antibody, the linker has an active reactive group that can react with certain amino acid residues, and the connection is achieved through the active reactive group. Thiol-specific reactive groups are preferred and include, for example, maleimides, halogenated amides (e.g., iodine, bromo, or chlorinated); halogenated esters (e.g., iodine, bromo, or chlorinated). ); Halogenated methyl ketones (e.g. iodine, bromo or chlorinated), benzyl halides (e.g. iodine, bromo or chlorinated); vinyl sulfone, pyridyl disulfide; mercury derivatives such as 3,6- Bis-(mercurymethyl)dioxane, and the counter ion is acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiosulfonate. catch The head may comprise, for example, a maleimide linked to the antibody via thiosuccinimide.

The drug can be any cytotoxic, cytostatic, or immunosuppressive drug. In embodiments, the linker connects the antibody and the drug, and the drug has a functional group that can form a bond with the linker. For example, the drug may have an amino, carboxyl, thiol, hydroxyl, or ketone group that can form a bond with the linker. In the case where the drug is directly attached to the linker, the drug has reactive groups before being attached to the antibody.

Useful drug classes include, for example, antitubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folate antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors , Catharanthus roseus alkaloids, etc. Examples of particularly useful classes of cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins (camptothecins), docarmycins/duocarmycins, etoposides, maytansines and maytansinoids (such as DM1 and DM4), taxanes ( taxanes), benzodiazepines or benzodiazepine containing drugs (such as pyrrolo[1,4]benzodiazepines (PBDs), indoline benzodiazepines (indolinobenzodiazepines) and oxazolidinobenzodiazepines (oxazolidinobenzodiazepines) and vinca alkaloids.

In the present invention, drug-linkers can be used to form ADCs in one simple step. In other embodiments, bifunctional linker compounds can be used to form ADCs in a two- or multi-step process. For example, a cysteine residue reacts with the reactive part of the linker in a first step, and in a subsequent step, the functional group on the linker reacts with the drug, forming an ADC.

Typically, functional groups on the linker are selected to facilitate specific reaction with appropriate reactive groups on the drug moiety. As a non-limiting example, azide-based moieties can be used to specifically react with reactive alkynyl groups on the drug moiety. The drug is covalently bound to the linker via a 1,3-dipolar cycloaddition between the azide and alkynyl groups. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides), isocyanates and isothiocyanates (suitable for reaction with amines) and activated esters, such as N-hydroxysuccinimide ester (suitable for reactions with amines and alcohols). These and other conjugation strategies, such as those described in Bioconjugation Technology, 2nd edition (Elsevier), are well known to those skilled in the art. Those skilled in the art can understand that for the selective reaction of a drug moiety and a linker, when a complementary pair of reactive functional groups is selected, each member of the complementary pair can be used for both the linker and the drug.

The present invention also provides a method for preparing an ADC, which may further include: combining an antibody with a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).

In certain embodiments, methods of the invention comprise conjugating an antibody to a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the methods of the invention further comprise: placing the antibody under conditions sufficient to covalently link the drug moiety to the antibody through the linker. The linker conjugate binds to the drug moiety.

application

The invention also provides the use of the bispecific cell engager molecules, antibody conjugates ADC, recombinant proteins, chimeric antigen receptor (CAR) constructs and/or immune cells of the invention, for example for the preparation of diagnostic preparations or preparations drug.

Preferably, the medicine is a medicine used to prevent and/or treat diseases related to abnormal expression or function of Pep42 receptor CD3.

In the present invention, the diseases related to abnormal expression or function of Pep42 receptor are conventional diseases related to abnormal expression or function of Pep42 receptor in this field. Preferably, the Pep42 receptor is GRP78, and the diseases related to abnormal expression or function of the Pep42 receptor include: tumors, aging, cardiovascular disease, obesity, or combinations thereof.

In the present invention, the cancer is a conventional cancer in this field, including hematological tumors and solid tumors, preferably pancreatic cancer or leukemia.

Testing purposes and kits

The bispecific cell engager molecules of the present invention or their ADCs can be used in detection applications, for example, in detecting samples to provide diagnostic information.

In the present invention, the samples (samples) used include cells, tissue samples and biopsy specimens. The term "biopsy" as used herein shall include all types of biopsies known to those skilled in the art. Biopsies used in the present invention may thus include, for example, resection samples of tumors, tissue samples prepared by endoscopic methods or puncture or needle biopsy of organs.

Samples used in the present invention include fixed or preserved cell or tissue samples.

The invention also provides a kit containing the antibody (or fragment thereof) of the invention. In a preferred embodiment of the invention, the kit further includes a container, instructions for use, a buffer, etc. In a preferred embodiment, the antibody of the present invention can be immobilized on a detection plate.

pharmaceutical composition

The invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein or its ADC or corresponding immune cell, and a pharmaceutically acceptable carrier. Generally, these materials may be formulated in a nontoxic, inert, and pharmaceutically acceptable aqueous carrier medium, usually at a pH of about 5-8, preferably at a pH of about 6-8, although the pH may vary. It will vary depending on the nature of the substance formulated and the condition to be treated.

The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to) intratumoral, intraperitoneal, intravenous, or topical administration. Typically, the pharmaceutical compositions of the present invention are administered The route of administration is preferably injection or oral administration. The injection administration preferably includes intravenous injection, arterial injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection. The pharmaceutical composition is in various conventional dosage forms in this field, preferably in the form of solid, semi-solid or liquid, and can be in the form of aqueous solution, non-aqueous solution or suspension, and more preferably in the form of tablets, capsules, granules , injections or infusions, etc.

The antibody of the present invention can also be expressed in cells from a nucleotide sequence and used for cell therapy. For example, the antibody can be used for chimeric antigen receptor T cell immunotherapy (CAR-T).

The pharmaceutical composition of the present invention is a pharmaceutical composition for preventing and/or treating diseases related to abnormal expression or function of Pep42 receptor and/or CD3.

The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned monoclonal antibody of the present invention (or its conjugate) and pharmaceutical acceptable carrier or excipient. Such carriers include, but are not limited to: saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The drug formulation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under sterile conditions. The active ingredient is administered in a therapeutically effective amount, for example, about 1 microgram/kg body weight to about 5 mg/kg body weight per day. Additionally, the polypeptides of the invention may be used with other therapeutic agents.

In a preferred embodiment of the present invention, the polypeptide of the present invention can be used in combination with other therapeutic agents for treating and/or preventing cancer and/or cancer metastasis.

In the present invention, preferably, the pharmaceutical composition of the present invention further includes one or more pharmaceutical carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in this field, and the pharmaceutical carrier can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipients are conventional pharmaceutical excipients in this field, preferably including pharmaceutically acceptable excipients, fillers or diluents. More preferably, the pharmaceutical composition includes 0.01 to 99.99% of the above-mentioned protein and 0.01 to 99.99% of the pharmaceutical carrier, and the percentage is the mass percentage of the pharmaceutical composition.

In the present invention, preferably, the dosage of the pharmaceutical composition is an effective amount, and the effective amount is an amount that can alleviate or delay the progression of diseases, degenerative or damaging conditions. The effective amount can be determined on an individual basis and will be based in part on considerations of the condition to be treated and the results sought. One skilled in the art can determine an effective amount by using such factors as the above on an individual basis and using no more than routine experimentation.

When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is generally at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 mg per kilogram of body weight, Preferably the dose is about 10 micrograms/kg body weight to about 20 mg/kg body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the skill of a skilled physician.

Methods and compositions for detecting Pep42 receptor CD3 in samples

The invention also provides a method for detecting Pep42 receptor and/or CD3 in a sample (for example, detecting overexpression of Pep42 receptor and CD3), which includes the following steps: contacting the above-mentioned antibody with the sample to be tested in vitro, and detecting the above-mentioned It only depends on whether the antibody combines with the sample to be tested to form an antigen-antibody complex.

The meaning of overexpression is conventional in the art, and refers to the overexpression of RNA or protein of Pep42 receptor and CD3 in the sample to be tested (due to increased transcription, post-transcriptional processing, translation, post-translational processing and protein degradation changes), and local overexpression and increased functional activity due to altered protein transport patterns (increased nuclear localization) (as in the case of increased enzymatic hydrolysis of the substrate).

In the present invention, the detection method of whether the above-mentioned combination forms an antigen-antibody complex is a conventional detection method in this field, preferably a flow cytometry test (FACS) detection.

The present invention provides a composition for detecting Pep42 receptor and CD3 in a sample, which includes the above-mentioned antibodies, recombinant proteins, antibody conjugates, immune cells, or combinations thereof as active ingredients. Preferably, it also includes a compound composed of the functional fragment of the above-mentioned antibody as an active ingredient.

Main advantages of the invention

The bispecific cell adapter molecule constructed by the present invention targets GRP78 tumor antigen and T cells at the same time, and is directly infused into the body, or carried by cells in the body (such as NK cells, T cells, CAR-T cells, etc.) and carried in the body Continuous expression of the antibody protein allows the bispecific cell adapter molecule to exert its killing effect in the body while being accompanied by a sufficient number of T effector cells, further optimizing the efficiency of the effect. Its main advantages include:

1) High targeting: Prepare bispecific cell adapter molecules targeting GRP78-positive tumors, which can effectively bind to the receptor sites of tumor target cells. Moreover, due to the cyclic peptide structure of PEP42, the bispecific cell adapter molecule of the present invention has stronger affinity with target cells, stronger binding, and more specific and stable killing.

2) Small molecular weight: The bispecific cell adapter of the present invention has an scFv structure at one end and a cyclic peptide structure with only 13 amino acids at the other end. It is not only conducive to binding tumor cells and T cell surface antigens in the blood, but also has a total protein molecular weight Small and easy to produce.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the invention and are not intended to limit the scope of the invention. Experimental methods without specifying specific conditions in the following examples usually follow conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to manufacturing Conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

The sequences involved in the embodiments of the present invention are shown in the table below.

Table 2 Sequence List

Among them, positions 1-894 of SEQ ID NO: 19 are the nucleotide coding sequence, and the last three bases TAG are the stop codons.

Example 1 Construction and expression of antibodies

1.1 Preparation of target gene

In this example, the sequence of a bispecific cell adapter molecule was constructed.

The PEP42 ligand sequence used in this example is derived from the GRP78 ligand (see Targeting heat shock proteins on cancer cells: selection, characterization and cell-penetrating properties of peptide GRP78 ligands) , characterization, and cell-penetrating properties of a peptidic GRP78ligand). Biochemistry, 2006.45(31):p.9434-44.).

The anti-CD3 antibody sequence used in this example is derived from the sequence of the antibody clone OKT3 (see Journal of Biochemistry, 1996, 120:657-662.). The VH and VL chains of OKT3 are shown in Figure 1. The source of the VH chain sequence of the anti-CD3 antibody sequence used in the present invention can be found in GenBank BAA11539.1, and the source of the VL chain sequence can be found in GenBank AAC28463.1.

The anti-CD3 antibody VH and VL are connected by linker2 (SEQ ID NO:13) to form the VH-linker2-VL single-chain structure of OKT3. This single-chain structure is collectively referred to as OKT3 below. Its amino acid sequence and nucleotide sequence are as shown in the table 2 shown.

Linker1 (SEQ ID NO:16) is used to connect PEP42 and OKT3 to construct the target gene of the present invention (PEP42-linker1-OKT3). Its structure is shown in Figure 2(A), and its full-length amino acid sequence and nucleotide sequence are as follows As shown in Table 2.

1.2 Plasmid construction

The plasmid was constructed using the lentiviral vector pCDH-CMV-MCS-EF1-Puro (see Myeloid Leukemia. Mol Ther, 2016.24(9):p.1615-26.). The target gene is synthesized by direct synthesis, and EcoRI and Swa I enzyme cutting sites and protective bases are added to its 3' end and 5' end respectively. After being digested with EcoR I and Swa I, it will be digested with EcoRI and Swa I. Swa I digested vector ligation. After comparing the sequencing results, competent E. coli (Stbl3) was transformed. All plasmids were extracted using QIAGEN's endotoxin-free plasmid extraction kit and digested with Hind III for identification. The identification results are shown in Figure 2(B).

1.3 Virus packaging

HEK-293T cells were cultured in 15 cm culture dishes for virus packaging. When the HEK-293T cell confluence is about 80%-90% for transfection, prepare 2ml OPTIMEM dissolved plasmid mixture (core plasmid 20ug, pCMVΔR8.9 10ug, PMD2.G 4ug); in another centrifuge tube, add 2ml OPTIMEM and lipo 8000 for 68ul. After standing at room temperature for 5 minutes, the plasmid complex was added to the liposome complex, and then left at room temperature for 20 minutes. The above mixture was added dropwise to HEK-293T cells, incubated at 37°C for 6 hours and then the medium was removed. Re-add pre-warmed complete medium. After collecting the virus supernatant at 48 hours and 72 hours, centrifuge at 3000 rpm at 4°C for 20 minutes. After filtering with a 0.45um filter membrane, centrifuge at 25000 rpm at 4°C for 2.5 hours to concentrate the virus. After the concentrated virus was dissolved in 30 ul of virus lysis solution overnight, the virus titer was detected by QPCR. The results showed that the virus titer met the requirements.

1.4 Protein preparation

Infect CHO cells with packaged lentivirus and select with puromycin for one week, as shown in Figure 2(C). After expanded culture, the supernatant was collected and separated using a Histidine tag fusion protein purification column. The loading speed was controlled at a constant flow of 1 ml/min, and then imidazole was used for stepwise gradient elution. Identification and analysis using Coomassie Brilliant Blue and Western Blot. The results are shown in Figure 2(D,E). Finally, a 0.22um filter membrane was used for filtration and sterilization for subsequent in vivo and in vitro experiments.

1.5 Construction of target cells carrying luciferase

The pTomo-CMV-Luciferase-IRES-Puro lentivirus packaging procedure is the same as in Example 1.2. The virus infected U937, KG-1A, K562, ASPC1, BXPC3, MHCC-97H, and PANC1 cells and then screened with Puromycin (1ug/ml) for 2 weeks. U937, KG-1A, K562, ASPC1, BXPC3, MHCC-97H, PANC1-luciferase cells.

Example 2 Detection of the binding ability of GRP78-CD3/BiTE to target cells or T cells

The binding of GRP78-CD3/BiTE and negative control CD19-CD3/BiTE to target cells or T cells was detected by flow cytometry. The cells were co-incubated with the above-mentioned bispecific cell adapter, then incubated with anti-His tag antibody, and finally incubated with FITC-conjugated goat anti-rabbit IgG (H+L) secondary antibody, and measured on a flow cytometer. Quantify the fluorescence of stained cells.

Results: Two GRP78-positive acute myeloid leukemia cell lines (U937 and KG-1A) and a GRP78-negative cell line K562 were tested. It was found that the binding ability of GRP78-CD3/BiTE to the three cell lines was 89.6% and 89.6%, respectively. 72.7% and 1.84% (Figure 3A). The negative control had no binding to the three cell lines. At the same time, the binding abilities of this adapter to pancreatic cancer solid tumor cell lines (ASPC-1, BXPC-3, MHCC-97H and PANC-1) were determined to be 90.3%, 79.1%, 85.2% and 5.74% respectively (Figure 3B ), consistent with the results of GRP78-positive cell lines. This result shows that the adapter can bind to GRP78-positive cells. T cells were isolated from peripheral blood of healthy people, and the binding ability of GRP78-CD3/BiTE and negative control to T cells was tested. It was found that both the adapter and the negative control could bind well to T cells (Figure 3C).

Example 3 Detection of target cell killing ability and cytokine secretion of T cells mediated by GRP78-CD3/BiTE

The above T cells were incubated with U937, KG-1A (GRP78 positive) leukemia cells and K562 (GRP78 negative) leukemia cells respectively at an effector-target ratio of 10:1 for 24 hours. The T cells were effector cells and the concentration was 2*10 ⁵ /mL, 100uL per well; leukemia cells are target cells. The white blood cells are labeled with CFSE and then added to the co-culture system. The concentration is 2*10 ⁴ /mL, 100uL per well, a total of 200uL per well (cell culture medium: advance 1640 medium ( Gibco)+10% fetal bovine serum (Gibco)+1% penicillin, streptomycin (Gibco)). An ELISA kit was used to detect the secretion of cytokines in the cell culture supernatants of 3 samples in each group.

The cell killing effect was detected by flow cytometry. After 24 hours, cells were collected, resuspended in 1mL PBS, stained with PI (1:1000), and flow cytometric detection was performed after staining for 10 minutes.

In solid tumor cells, T cells were incubated with ASPC1, BXPC3, MHCC-97H (GRP78 positive) solid tumor cells and PANC1 (GRP78 negative) solid tumor cells respectively at an effect-to-target ratio of 10:1 for 24 hours, in which T cells were Effector cells, the concentration is 2*10 ⁴ /mL, 100uL per well; tumor cells are target cells, the concentration is 2*10 ³ /mL, 100uL per well, a total of 200uL per well (cell culture medium: advance 1640 medium (Gibco) +10% fetal bovine serum (Gibco) +1% penicillin, streptomycin (Gibco)).

The killing effect of ASPC1, BXPC3, and PANC1 solid tumor cells was detected using promega fluorescence detection kit. First, cells were treated with 30ul of 1*PLB lysis buffer for 20 minutes, then 30ul of substrate was added to each well and immediately detected with a BioTek microplate reader. Cell killing % = (1-fluorescence value of target cells when containing effector cells/fluorescence value of target cells when there are no effector cells)×100%.

Results: As shown in Figure 4, this adapter mediates T cell cytotoxicity against GRP78-expressing U937 and KG-1A cells in a dose-dependent manner, but does not exhibit killing ability against GRP78-negative K562 cells (Figure 4A). The release amounts of cytokines (TNF-α, IFN-γ and GZMB) in the above co-culture system are also consistent with the corresponding killing effects (Figure 5). At the same time, similar killing results were also obtained in solid tumor cell lines (Figure 4B).

Example 4 Detection of GRP78-CD3/BiTE-mediated T cell activation ability in PBMC

PBMC were incubated with U937, KG-1A (GRP78 positive) leukemia cells and K562 (GRP78 negative) leukemia cells respectively at an effect-to-target ratio of 10:1 for 72 hours. T cells were effector cells at a concentration of 2*10 ⁵ /mL. , 100uL per well; leukemia cells are target cells, the concentration is 2*10 ⁴ /mL, 100uL per well, a total of 200uL per well (the cell culture medium is the same as in Example 3).

The cell killing effect was detected by flow cytometry. After 72 hours, cells were collected, incubated with CD3/CD25/CD69 flow cytometry antibodies and then detected.

Results: As shown in Figure 6, GRP78-CD3/BiTE can mediate the killing of GRP78-expressing cells by PBMC. In a co-culture system of PBMC cells and acute myeloid leukemia cells, GRP78-CD3/BiTE can effectively activate T cells to express CD69 and CD25 only in the presence of GRP78-positive cell lines (Figure 6B, C), indicating that this adapter GRP78-CD3/BiTE mediates and activates T cells to produce killing.

Example 5 GRP78-CD3/BiTE clears human acute myeloid leukemia in xenograft mouse model

Inject 1.0 x 10 ⁶ U937-EGFP-Luci or KG1a-Luci cells intravenously into NCG mice through the tail vein. After randomization, mice were intravenously injected with T cells. Different concentrations (0.004mg/kg, 0.2mg/kg, 1mg/kg) of bispecific antibodies were injected into the tail vein, repeated every other day. Body weight and tumor volume were measured every 3 days. and quantify tumor progression through serial bioluminescence imaging. Bioluminescence images were captured by an IVIS imaging system and quantified using Living Image software 4.1 (PerkinElmer). Mice were sacrificed when they died or developed symptoms of hind limb paralysis or at the end of the experimental time point.

Results: By tracking tumor growth, it was found that compared with the NTD group that only injected T cells, the group that received the negative control CD19-CD3/BiTE had no significant effect on tumor growth. 1 mg/kg and 0.2 mg/kg GRP78-CD3/BiTE high-dose and medium-dose groups could significantly inhibit tumor growth, and the low-dose group slowed down the progression of tumors (Figure 7B, C), accompanied by the cytokine TNF in mice. - Increase in alpha levels (Fig. 7D). In the T cells alone and the negative control group, the first signs of the disease, such as reduced mobility, hypothermia and thinning hair, appeared within 15 days of tumor formation. The cell line progressed very rapidly.

Example 6 GRP78-CD3/BiTE mediates T cell killing of primary acute myeloid leukemia

To determine whether this adapter mediates T cell cytotoxicity against primary AML cells. At a concentration of GRP78-CD3/BiTE 100ng/ml, tumor cells and T cells in primary AML blood samples were co-cultured for 24 hours and then their cytotoxicity was measured. The results showed that the adapter had significant cytotoxicity against CD34-positive and GRP78-positive primary AML tumor cell samples in 3 patients (Figure 8B, E, H). Compared with the control group, the level of cytokine TNF-α released in the supernatant of the experimental group was significantly higher, indicating that this adapter mediates T cells to specifically trigger immune cytotoxicity of patients' AML cells (Figure 8C,F ,I). In addition, immunofluorescence staining was performed on the above-mentioned primary AML tumor cell samples, and it was confirmed that the AML patient samples with killing effect were enriched in GRP78 on the plasma membrane (Figure 8D, G, J).

All documents mentioned in this application are incorporated by reference in this application to the same extent as if each individual document was individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

A cell adapter molecule, characterized in that the cell adapter molecule includes:

(a) a first binding domain, the first binding domain specifically binds to the Pep42 receptor, and the first binding domain has a cyclic peptide structure; and

(b) A second binding domain that specifically binds CD3.
The cell binder molecule of claim 1, wherein the first binding domain is derived from a Pep42 ligand peptide, and the Pep42 ligand peptide has SEQ ID NO: 2 (CTVALPGGYVRVC) the sequence of.
The cell binder molecule of claim 1, wherein the second binding domain has a peptide segment derived from an anti-human CD3 antibody.
The cell binder molecule of claim 1, wherein the second binding domain is an anti-CD3 antibody, the antibody includes a VH segment, and the VH segment has the following complementarity determining region CDR:

VH-CDR1 shown in SEQ ID NO:5,

VH-CDR2 shown in SEQ ID NO:6, and

VH-CDR3 shown in SEQ ID NO:7;

And the antibody includes a VL segment, and the VL segment has the following complementarity determining region CDR:

VL-CDR1 shown in SEQ ID NO:8,

VL-CDR2 shown in SEQ ID NO:9, and

VL-CDR3 shown in SEQ ID NO:10;

Furthermore, any amino acid sequence in the above-mentioned CDR sequence also includes a derivative antibody composed of a heavy chain and a light chain, optionally adding, deleting, modifying and/or substituting at least one amino acid, so as to contain the derivative CDR sequence. Derived sequences capable of retaining CD3 binding affinity.
The cell binder molecule of claim 1, wherein the cell binder molecule has a structure selected from the following formula (I) or (II) from the N-terminus to the C-terminus:
SD 1 -L 1 -D 2 -T (I); and
SD 2 -L 1 -D 1 -T (II),

In the formula,

Each "-" is independently a connecting peptide or peptide bond;

S is none or signal peptide sequence;

D 1 is the first binding domain;

L 1 is none or the first connecting peptide;

D 2 is the second binding domain;

T is None or Tagged protein.
The cell binder molecule of claim 5, wherein the amino acid sequence of D1 is as shown in SEQ ID NO:2; or has a sequence identity of ≥85% with SEQ ID NO:2, preferably ≥ 90%, preferably ≥93%, or have a difference of 1, 2 or 3 amino acids compared with SEQ ID NO:2, and have the same or similar function as the sequence shown in SEQ ID NO:2.
The cell adapter molecule of claim 1, wherein the amino acid sequence of the bispecific cell adapter molecule is selected from:

(i) The amino acid sequence shown in SEQ ID NO: 20;

(ii) On the basis of the sequence shown in SEQ ID NO:20, one or more amino acid residues are replaced, deleted, changed or inserted, or 1 to 30 amino acid residues are added to its N-terminal or C-terminal , preferably 1 to 10 amino acid residues, more preferably 1 to 5 amino acid residues, thereby obtaining an amino acid sequence; and the obtained amino acid sequence has ≥85% with the sequence shown in SEQ ID NO:20 (preferably ≥90%, more preferably ≥95%, such as ≥96%, ≥97%, ≥98% or ≥99%) sequence identity; and the obtained amino acid sequence is identical to the sequence shown in (i) Have the same or similar functions.
A recombinant protein, characterized in that the recombinant protein includes the cell adapter molecule according to claim 1.
A polynucleotide, characterized in that the polynucleotide encodes a polypeptide selected from the group consisting of:

(1) The bispecific cell adapter molecule of claim 1; and

(2) The recombinant protein according to claim 8.
A vector, characterized in that the vector contains the polynucleotide of claim 9.
An engineered host cell, characterized in that the host cell contains the vector according to claim 10 or the polynucleotide according to claim 9 is integrated into the genome.
A pharmaceutical composition, characterized in that the pharmaceutical composition contains:

(i) an active ingredient selected from the group consisting of: a cell adapter molecule as claimed in claim 1, a recombinant protein as claimed in claim 8, a host cell as claimed in claim 11, or a combination thereof ;as well as

(ii) One or more pharmaceutically acceptable carriers, diluents, fillers, binding agents, excipients, or combinations thereof.
The cell adapter molecule according to claim 1, the recombinant protein according to claim 8, the host cell according to claim 11, and/or the pharmaceutical composition according to claim 12 is prepared for use in treatment. Use in medicines for diseases associated with abnormal expression or function of Pep42 receptors.
The use according to claim 13, wherein the Pep42 receptor is csGRP78.
The use according to claim 14, wherein the disease is a malignant tumor overexpressing csGRP78.