CN115991786A

CN115991786A - Compositions for targeted MSLN immune cell therapies

Info

Publication number: CN115991786A
Application number: CN202211253555.8A
Authority: CN
Inventors: 李志远; 张振清; 黄威峰; 贾云莉; 曾竣玮
Original assignee: Pumis Biotechnology Suzhou Co ltd
Current assignee: Pumis Biotechnology Suzhou Co ltd
Priority date: 2021-10-18
Filing date: 2022-10-13
Publication date: 2023-04-21

Abstract

The present invention relates to the field of immunotherapy, in particular, the present invention provides compositions for targeted MSLN immune cell therapies involving the combined expression of chimeric CD3 fusion proteins and dual-specific T cell activating elements (bitas) against CD3 and mesothelin, and engineered immune cells expressing the chimeric CD3 fusion proteins and dual-specific T cell activating elements.

Description

Compositions for targeted MSLN immune cell therapies

Technical Field

Background

Tumor immunotherapy, which is a therapeutic method for combating tumors by mobilizing the immune system of the body, has become the mainstream therapy of tumors. Currently common tumor immunotherapy is: t cell targeted immune checkpoint inhibitors (anti-PD-1/PD-L1/CTLA-4 antibodies), cell therapy (CAR-T, TCR-T), CD3 targeted bispecific antibodies, and the like. However, only new anti-PD-1/PD-L1 antibodies are marketed for clinical treatment of solid tumors, and the tumor response rate is generally lower than 30%, so that the clinical efficacy of the solid tumor immunotherapy still has a great room for improvement.

Targeting Mesothelin (MSLN) -positive solid tumor cell therapies is one of the potential development directions. Mesothelin (MSLN) is a 40kD size glycosyl phosphatidylinositol linked (GPI) glycoprotein found on the surface of mesothelial cells, mesothelioma, epithelial ovarian cancer and some squamous cell carcinoma. The 69kD precursor is synthesized first, and then through enzymolysis, 30kD N-terminal secretion type and 40kD GPI connection membrane combination type are obtained. Although mesothelin is also expressed in normal cells and is not a tumor-specific antigen, mesothelin is still a very potential target for immunotherapy because it is expressed at significantly higher levels in various tumor cells than in normal cells. Mesothelin is highly expressed in about 30% of tumors compared to low expression levels in normal cells, including: epithelial-like intercostal carcinoma, pancreatic adenocarcinoma, ovarian serous carcinoma, head and neck squamous carcinoma, nasal carcinoma, gastric carcinoma, cholangiocarcinoma, endometrial carcinoma, triple-negative breast carcinoma, and other tumors with different differentiation degrees.

Targeting mesothelin CAR-T therapy and TCR-T therapy has been reported. First-stage clinical data targeting mesothelin CAR-T for treatment of malignant solid tumors were reported by Andrew r.haas et al, 11 in 2019, which showed that MSLN CAR-T treatment was well tolerated overall, but had limited efficacy, with no significant tumor shrinkage observed (Haas, andrew R et al, "Phase I Study of Lentiviral-Transduced Chimeric Antigen Receptor-Modified T Cells Recognizing Mesothelin in Advanced Solid cancer," Molecular therapy: the journal of the American Society of Gene Therapy vol.27,11 (2019): 1919-1929).

7.27.2020, TCR ² Therapeutic applications published phase 1 clinical data targeting MSLN cell therapy product TC-210, wherein TC-210 recognizes that tumor antigen MSLN can exert an anti-tumor effect by activating its TCR signaling pathway, but cannot activate unedited T cells or tumor infiltrating T cells to exert an anti-tumor effect. In terms of safety, cytokine storm (CRS) occurred in 3 cases, with 1 in 2 cases and 3 in 1 case. Grade 3 patients with CRS developed grade 3 pneumonia side effects after receiving Tocilizumab and hormonal therapy. The patient was considered to be unrelated to TC-210 by fungal sepsis at 34 days post-treatmenthttps:// investors.tcr2.com/events-and-presentations/presentations)。

As mentioned above, there is no report of significant efficacy of CAR-T cell therapies against solid tumors. TIL and TCR-T therapies have been reported to be therapeutic, but since the clinical application of TCR-T cell therapies is HLA-restricted, most tumor-associated antigen positive patients cannot be used; the preparation process of the TIL is complex, and many tumor patients have fewer T cells infiltrated in tumor tissues, so that the separation success rate of the TIL is lower. This limits the practical use of TIL or TCR-T therapy.

Therefore, there is an urgent need to develop cell therapy products that can be prepared as simply as CAR-T production processes and can achieve significant therapeutic effects in clinical treatment of solid tumors.

Disclosure of Invention

The efficacy of TIL or TCR-T cell therapy in solid tumors is greatly different from that of CAR-T cell therapy, presumably due to the differential signaling of activated T cells. TIL or TCR-T mediates T cell killing of tumors by activating endogenous TCR signaling, and CAR-T mediates synthetic signaling of Chimeric Antigen Receptors (CARs). Based on the CAB-T (chireric CD3e and anti-CD3 based Bispecific T cell activator engineered T cells) technology disclosed in the inventors' prior patent application (PCT/IB 2020/058302), the inventors of the present application provided an Mesothelin (MSLN) -targeted immune cell therapy, MSLN CAB-T. The MSLN CAB-T provided by the application can improve the killing efficiency of the traditional targeted MSLN immune cell therapy by realizing the organic integration of the CAR signal, the TCR signal and the targeted MSLN-CD3 bispecific antibody activity, thereby hopefully solving the treatment bottleneck of the current cell therapy aiming at the solid tumor and achieving important breakthrough in the clinical treatment of the future solid tumor.

Bispecific antibody constructs

Thus, in a first aspect, the present application provides a bispecific antibody construct comprising a first binding domain that specifically binds MSLN and a second binding domain that specifically binds CD 3; wherein the first binding domain is a single domain antibody or a tandem or oligomeric form thereof, the single domain antibody comprising:

(a) CDR1, consisting of the sequence: SEQ ID NO. 2, or a sequence having a substitution, deletion or addition of one or several amino acids as compared to it (e.g.a substitution, deletion or addition of 1, 2 or 3 amino acids);

(b) CDR2, consisting of the sequence: SEQ ID NO. 3, or a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto;

(c) CDR3 consisting of the sequence: SEQ ID NO. 4, or a sequence having one or several amino acid substitutions, deletions or additions (e.g.1, 2 or 3 amino acid substitutions, deletions or additions) as compared thereto.

In certain preferred embodiments, the first binding domain comprises a CDR1 as shown in SEQ ID NO. 2, a CDR2 as shown in SEQ ID NO. 3, and a CDR3 as shown in SEQ ID NO. 4.

In certain embodiments, the CDRs contained in the single domain antibody are preferably determined by the IMGT numbering system.

In certain embodiments, the first binding domain comprises a sequence as set forth in SEQ ID NO. 1, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) as compared to SEQ ID NO. 1.

In certain embodiments, the first binding domain comprises the amino acid sequence set forth in SEQ ID NO. 1.

In certain embodiments, the second binding domain is selected from an antibody or antigen binding fragment thereof that specifically binds CD 3.

In certain embodiments, the antigen binding fragment is selected from a single domain antibody, a single chain antibody, a Fab, or a tandem or oligomeric form thereof.

In certain embodiments, the second binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) of a monoclonal antibody selected from the group consisting of: L2K, UCHT, OKT, F6A, SP34.

In certain embodiments, the second binding domain is humanized.

In certain embodiments, the second binding domain comprises a VH as set forth in SEQ ID NO. 5, and/or a VL as set forth in SEQ ID NO. 6.

In certain embodiments, the second binding domain comprises the amino acid sequence set forth in SEQ ID NO. 7.

In certain embodiments, the first binding domain and/or the second binding domain of the bispecific antibody construct has cross-species specificity (e.g., has cross-species specificity for humans, monkeys, and mice). In certain embodiments, the first binding domain and/or the second binding domain of the bispecific antibody construct has trans-species specificity for a member of the mammalian class of primates.

In certain embodiments, the first binding domain is linked to the N-terminus and/or the C-terminus (e.g., N-terminus) of the second binding domain, optionally via a peptide linker.

In certain embodiments, the first binding domain is linked to the N-terminus of the second binding domain by a peptide linker.

In certain embodiments, the peptide linker is a rigid or flexible linker, e.g., a peptide linker comprising one or more glycine and/or one or more serine.

In certain embodiments, the peptide linker comprises the amino acid sequence set forth in SEQ ID NO. 8.

In certain embodiments, the bispecific antibody construct comprises the amino acid sequence as shown in SEQ ID NO. 9.

In certain embodiments, the bispecific antibody construct further comprises a signal peptide and/or a detectable label (e.g., a tag protein). In certain embodiments, the detectable label is selected from the group consisting of tag proteins. Tag proteins are well known in the art, examples of which include, but are not limited to His, flag, GST, MBP, HA, myc or biotin, etc., and it is known to those skilled in the art how to select an appropriate tag protein according to the desired purpose (e.g., purification, detection, or labeling).

In certain embodiments, the bispecific antibody construct comprises a signal peptide at its N-terminus.

In certain embodiments, the bispecific antibody construct comprises a detectable label at its C-terminus.

In a second aspect, the present application also provides an isolated nucleic acid molecule encoding a bispecific antibody construct as described above.

In a third aspect, the present application also provides a vector comprising an isolated nucleic acid molecule as described above.

In certain embodiments, the vector is a cloning vector or an expression vector. In certain embodiments, the vector is a lentiviral, adenoviral or retroviral vector.

In a fourth aspect, the present application also provides a host cell comprising an isolated nucleic acid molecule or vector as described above.

In certain embodiments, the host cell is an engineered immune cell.

In certain embodiments, the engineered immune cell secretes and expresses a bispecific antibody construct as described above.

In certain embodiments, the engineered immune cell is a T cell, NK cell, γδ T cell, NKT cell, or any combination thereof.

Composition and method for producing the same

In a fifth aspect, the present application also provides a composition comprising a bispecific antibody construct as described above, and a chimeric CD3 fusion protein, wherein the chimeric CD3 fusion protein comprises a polypeptide binding domain comprising a polypeptide recognizable by a second binding domain of the bispecific antibody construct; optionally one or more of the following domains:

(a) A joint or hinge region;

(b) A transmembrane domain;

(c) Costimulatory signaling domains

(d) A CD3 signaling activation domain;

in certain embodiments, the chimeric CD3 fusion protein comprises the polypeptide binding domain and a transmembrane domain from N-terminus to C-terminus. In certain embodiments, the chimeric CD3 fusion protein further comprises a costimulatory signaling domain and/or a CD3 signaling activation domain at the C-terminus of the transmembrane domain. In certain embodiments, the chimeric CD3 fusion protein further comprises a linker or hinge region between the polypeptide binding domain and the transmembrane domain.

In certain embodiments, the chimeric CD3 fusion protein comprises, in order from N-terminus to C-terminus, the polypeptide binding domain, a linker or hinge region, a transmembrane domain, a costimulatory signaling domain, and a CD3 signaling domain.

In certain embodiments, the polypeptide that is recognizable by the second binding domain comprises or consists of a CD3e extracellular region or fragment thereof.

In certain embodiments, the polypeptide recognizable by the second binding domain comprises or consists of amino acids 1-27 of the CD3e protein.

In certain embodiments, the polypeptide recognizable by the second binding domain comprises or consists of amino acids 1-104 of the CD3e protein.

In certain embodiments, the polypeptide that is recognized by the second binding domain comprises a sequence as set forth in SEQ ID NO. 11.

In certain embodiments, the hinge region is a hinge region selected from the group consisting of: CD8, CD28, 4-1BB, or any combination thereof.

In certain embodiments, the hinge region is a hinge region of CD 8. In certain embodiments, the hinge region comprises or consists of the amino acid sequence set forth in SEQ ID NO. 12.

In certain embodiments, the transmembrane domain is a transmembrane domain selected from the group consisting of: CD8, CD28, CD3, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134,4-1BB, CD154, or any combination thereof.

In certain embodiments, the transmembrane domain is the transmembrane domain of CD 8. In certain embodiments, the transmembrane domain comprises or consists of the amino acid sequence shown in SEQ ID NO. 13.

In certain embodiments, the costimulatory signaling domain is an intracellular domain selected from the group consisting of: CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134,4-1BB, PD1, DAPLO, CDS, ICAM-1, LFA-1 (CDLLA/CD 18), ICOS (CD 278), NKG2D, GITR, TLR2, or any combination thereof.

In certain embodiments, the costimulatory signaling domain is the intracellular domain of 4-1 BB. In certain embodiments, the costimulatory signaling domain comprises or consists of the amino acid sequence depicted as SEQ ID NO. 14.

In certain embodiments, the CD3 signaling activation domain is a cd3ζ intracellular domain. In certain embodiments, the CD3 signal activation domain comprises or consists of the amino acid sequence set forth in SEQ ID NO. 15.

In certain embodiments, the chimeric CD3 fusion protein comprises the amino acid sequence set forth in SEQ ID NO. 16.

In certain embodiments, the chimeric CD3 fusion protein further comprises a signal peptide and/or a detectable label (e.g., a tag protein).

In certain embodiments, the chimeric CD3 fusion protein comprises a signal peptide at its N-terminus.

In certain embodiments, the chimeric CD3 fusion protein comprises a detectable label at its C-terminus.

In a sixth aspect, the present application also provides an isolated nucleic acid molecule encoding a bispecific antibody construct as defined in the first aspect and a chimeric CD3 fusion protein as defined in the fifth aspect.

In certain embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding the bispecific antibody construct and a second nucleotide sequence encoding the chimeric CD3 fusion protein.

In certain embodiments, the first nucleotide sequence and the second nucleotide sequence are present on different isolated nucleic acid molecules.

In certain embodiments, the first nucleotide sequence and the second nucleotide sequence are present on the same isolated nucleic acid molecule in any order. In certain embodiments, the first nucleotide sequence and the second nucleotide sequence are linked in any order by a nucleotide sequence encoding a self-cleaving peptide (e.g., T2A). In certain embodiments, the first nucleotide sequence and the nucleotide sequence encoding a self-cleaving peptide and/or the second nucleotide sequence and the nucleotide sequence encoding a self-cleaving peptide optionally further comprise a nucleotide sequence encoding a peptide linker (e.g., GSG).

In certain embodiments, the first nucleotide sequence and/or the second nucleotide sequence optionally further comprises a nucleotide sequence encoding a signal peptide at its 5' end.

In a seventh aspect, the present application also provides a vector comprising the isolated nucleic acid molecule of the sixth aspect.

In certain embodiments, the vector comprises a first nucleotide sequence encoding the bispecific antibody construct and a second nucleotide sequence encoding the chimeric CD3 fusion protein, wherein the first nucleotide sequence and the second nucleotide sequence are located in the same expression cassette in any order.

In certain embodiments, the first nucleotide sequence and the second nucleotide sequence are linked in any order by a nucleotide sequence encoding a self-cleaving peptide (e.g., T2A). In certain embodiments, the first nucleotide sequence and the nucleotide sequence encoding a self-cleaving peptide and/or the second nucleotide sequence and the nucleotide sequence encoding a self-cleaving peptide optionally further comprise a nucleotide sequence encoding a peptide linker (e.g., GSG).

In certain embodiments, the vector comprises a first nucleotide sequence encoding the bispecific antibody construct and a second nucleotide sequence encoding the chimeric CD3 fusion protein, wherein the first nucleotide sequence and the second nucleotide sequence are located in different expression cassettes.

In certain embodiments, the first expression cassette comprising the first nucleotide sequence is located on the same vector or a different vector than the second expression cassette comprising the second nucleotide sequence.

In an eighth aspect, the present application also provides an engineered immune cell comprising the isolated nucleic acid molecule of the sixth aspect, or the vector of the seventh aspect.

In certain embodiments, the engineered immune cell expresses a chimeric CD3 fusion protein as defined in the fifth aspect and a bispecific antibody construct as defined in the first aspect.

In certain embodiments, the engineered immune cell secretly expresses the bispecific antibody construct.

In certain embodiments, the engineered immune cell expresses the chimeric CD3 fusion protein on its surface.

In a ninth aspect, the present application also provides an immune cell composition comprising the host cell of the fourth aspect and/or the engineered immune cell of the eighth aspect.

In certain embodiments, the immune cell composition further comprises an unedited and/or unsuccessfully edited immune cell.

In certain embodiments, the engineered immune cell count comprises at least 10% of the total number of cells of the immune cell composition.

In a tenth aspect, the present application also provides a method of making a host cell of the fourth aspect, comprising: (1) providing an immune cell; (2) Introducing the isolated nucleic acid molecule of the second aspect or the vector of the third aspect into the immune cell. The present application also provides a method of preparing the engineered immune cell of the eighth aspect, comprising: (1) providing an immune cell; (2) Introducing the isolated nucleic acid molecule of the sixth aspect or the vector of the seventh aspect into the immune cell.

In certain embodiments, the immune cells are selected from T cells, NK cells, γδ T cells, NKT cells, or any combination thereof.

In certain embodiments, in step (1), the immune cells are pre-treated, including sorting, activation and/or proliferation of immune cells.

In certain embodiments, the nucleic acid molecule or vector is introduced into the host cell in step (2) by viral infection or by transfection with a non-viral vector.

In certain embodiments, the step (2) is followed by a step of expanding the immune cells obtained in step (2).

In an eleventh aspect, the present application also provides a composition comprising the bispecific antibody construct of the first aspect, and an immune cell expressing a chimeric CD3 fusion protein; wherein the chimeric CD3 fusion protein is as defined in the fifth aspect.

In certain embodiments, the bispecific antibody construct is secreted by the immune cell or exogenous relative to the immune cell.

In a twelfth aspect, the present application also provides a pharmaceutical composition comprising the bispecific antibody construct of the first aspect, the host cell of the fourth aspect, the composition of the fifth aspect, the isolated nucleic acid molecule of the second or sixth aspect, the vector of the third or seventh aspect, the engineered immune cell of the eighth aspect, the immune cell composition of the ninth aspect or the composition of the eleventh aspect.

In certain embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or excipient.

In certain embodiments, the pharmaceutical composition comprises the host cell of the fourth aspect, the engineered immune cell of the eighth aspect, or the immune cell composition of the ninth aspect. In certain embodiments, the cells in the pharmaceutical compositionIs 1×10 in concentration ³ -1×10 ⁸ Individual cells/ml, preferably 1X 10 ⁴ -1×10 ⁷ Individual cells/ml.

In a thirteenth aspect, the present application also provides the use of a bispecific antibody construct of the first aspect, a host cell of the fourth aspect, a composition of the fifth aspect, an isolated nucleic acid molecule of the second or sixth aspect, a vector of the third or seventh aspect, an engineered immune cell of the eighth aspect, an immune cell composition of the ninth aspect, a composition of the eleventh aspect, or a pharmaceutical composition of the twelfth aspect for the preparation of a medicament for the prevention and/or treatment of a tumor in a subject.

In certain embodiments, the tumor is a MSLN positive tumor. Tumor types expressing MSLN are known to those skilled in the art, see, for example, weidmann S, gagelmann P, gorboson N, et al Mesothelin Expression in Human Tumors: A Tissue Microarray Study on 12,679 Tumors.Biomedicines.2021;9 (4):397.

In certain embodiments, the tumor is selected from solid tumors, such as mesothelioma, ovarian cancer, pancreatic cancer, sarcoma, lung adenocarcinoma, endometrial cancer, gastric adenocarcinoma, esophageal cancer, colorectal cancer, breast cancer, small intestine adenocarcinoma, ampulla cancer, uterine sarcoma, parotid adenoma, anal canal cancer, vaginal cancer, throat cancer, head and neck squamous carcinoma, nasal cancer, bile duct cancer, or cervical cancer.

In certain embodiments, the subject is a mammal, e.g., a human.

In certain embodiments, the bispecific antibody construct, isolated nucleic acid molecule, vector, host cell, composition, isolated nucleic acid molecule, vector, engineered immune cell, immune cell composition, or pharmaceutical composition is used alone or in combination with another pharmaceutically active agent (e.g., an antineoplastic agent).

In certain embodiments, the bispecific antibody construct, isolated nucleic acid molecule, vector, host cell, composition, isolated nucleic acid molecule, vector, engineered immune cell, immune cell composition, or pharmaceutical composition is used in combination with dasatinib, e.g., administered simultaneously or sequentially.

In a fourteenth aspect, the present application also provides a method for preventing and/or treating a tumor in a subject, comprising: administering to a subject in need thereof an effective amount of a bispecific antibody construct of the first aspect, an isolated nucleic acid molecule of the second aspect, a vector of the third aspect, a host cell of the fourth aspect, a composition of the fifth aspect, an isolated nucleic acid molecule of the sixth aspect, a vector of the seventh aspect, an engineered immune cell of the eighth aspect, an immune cell composition of the ninth aspect, a composition of the eleventh aspect, or a pharmaceutical composition of the twelfth aspect.

In certain embodiments, the method comprises administering to the subject an effective amount of the host cell of the fourth aspect, the engineered immune cell of the eighth aspect, or the immune cell composition of the ninth aspect.

In certain embodiments, the method further comprises administering to the subject an additional pharmaceutically active agent (e.g., an antineoplastic agent), e.g., simultaneously or sequentially.

In certain embodiments, the method further comprises administering dasatinib to the subject, e.g., simultaneously or sequentially.

In certain embodiments, the method further comprises administering to the subject a cytokine, pharmaceutical compound, or combination thereof that stimulates secretion of cells to enhance immune cell response capability.

In certain embodiments, the tumor is a MSLN positive tumor.

In certain embodiments, the subject is a mammal, e.g., a human.

The bispecific antibody construct of the first aspect, the isolated nucleic acid molecule of the second aspect, the vector of the third aspect, the host cell of the fourth aspect, the composition of the fifth aspect, the isolated nucleic acid molecule of the sixth aspect, the vector of the seventh aspect, the engineered immune cell of the eighth aspect, the immune cell composition of the ninth aspect, the composition of the eleventh aspect, or the pharmaceutical composition of the twelfth aspect may be formulated into any dosage form known in the medical arts, e.g., tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injectable solutions, injectable sterile powders and injectable concentrated solutions), inhalants, sprays, etc., preferably sterile and stable under the conditions of manufacture and storage. The preferred dosage form depends on the intended mode of administration and therapeutic use. One preferred dosage form is an injection. Such injections may be sterile injectable solutions.

The bispecific antibody construct of the first aspect, the isolated nucleic acid molecule of the second aspect, the vector of the third aspect, the host cell of the fourth aspect, the composition of the fifth aspect, the isolated nucleic acid molecule of the sixth aspect, the vector of the seventh aspect, the engineered immune cell of the eighth aspect, the immune cell composition of the ninth aspect, the composition of the eleventh aspect, or the pharmaceutical composition of the twelfth aspect may be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic, inguinal, intravesical, topical (e.g., powder, ointment, or drops), or nasal route. However, for many therapeutic uses, the preferred route/mode of administration is parenteral (e.g., intravenous injection or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled artisan will appreciate that the route and/or mode of administration will vary depending on the intended purpose. In certain embodiments, administration by intravenous injection or bolus injection is preferred.

Definition of terms

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Moreover, the virology, biochemistry, immunology laboratory procedures used herein are all conventional procedures widely used in the corresponding field. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

When used herein, the terms "for example," such as, "" including, "" comprising, "or variations thereof, are not to be construed as limiting terms, but rather as meaning" but not limited to "or" not limited to.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

As used herein, the term "antibody construct" refers to a molecule whose structure and/or function is based on the structure and/or function of an antibody. Thus, the antibody construct is capable of binding to its specific target or antigen. The antibody constructs according to the invention comprise the minimum structural requirements of the antibody that allow binding of the target. This minimum requirement can be defined, for example, by the presence of at least three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VH region) and/or three light chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VL region).

As used herein, the term "bispecific antibody construct" refers to a bispecific antibody construct that specifically binds to two antigen structures via different binding domains. The binding domains comprise the minimum structural requirements of the antibody based, including, for example, single domain antibodies, monoclonal antibodies, recombinant antibodies, chimeric antibodies, deimmunized antibodies, humanized antibodies, and human antibodies, that allow binding of the target. The binding domain may be a full length form of the antibody on which it is based or an antigen binding fragment thereof, e.g. the binding domain is selected from a single domain antibody, a single chain antibody, a Fab or a tandem or oligomeric form thereof. Thus, in certain embodiments, the binding domain may be monovalent, bivalent, or multivalent.

Furthermore, the bispecific antibody construct comprises a molecule consisting of only one polypeptide chain and a molecule consisting of more than one polypeptide chain, which chains may be identical (homodimer, homotrimer or homooligomer) or different (heterodimer, heterotrimer or hetero-oligomer).

Herein, the expression "single domain antibody, single chain antibody or oligomeric form of Fab" refers to a molecule consisting of two or more single domain antibodies, single chain antibodies or single Fab as repeat units; the expression tandem form of an antibody refers to a molecule formed by tandem of two or more single domain antibodies, single chain antibodies or single Fab.

As used herein, the term "antibody" refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains, each pair having one Light Chain (LC) and one Heavy Chain (HC). Antibody light chains can be classified as kappa (kappa) and lambda (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotypes of antibodies are defined as IgM, igD, igG, igA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also comprises a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CH 1, CH2 and CH 3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant domains are not directly involved in binding of antibodies to antigens, but exhibit a variety of effector functions, such as may mediate binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). VH and VL regions can also be subdivided into regions of high variability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FR). Each V is _H And V _L By the following sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 consist of 3 CDRs and 4 FRs arranged from amino-terminus to carboxy-terminus. The variable regions (VH and VL) of each heavy/light chain pair form antigen binding sites, respectively. The allocation of amino acids in various regions or domains may follow Kabat, sequences of Proteins of Immunological Interest (National Institutes of Health, bethesda, md. (1987 and 1991)), or Chothia&Lesk (1987) J.mol.biol.196:901-917; chothia et al (1989) Nature 342:878-883.

As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in an antibody variable region that are responsible for antigen binding. Three CDRs, designated CDR1, CDR2 and CDR3, are contained in each of the variable regions of the heavy and light chains. The precise boundaries of these CDRs may be defined according to various numbering systems known in the art, e.g., as in the Kabat numbering system (Kabat et al, sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md, 1991), the Chothia numbering system (Chothia & Lesk (1987) J.mol. Biol.196:901-917; chothia et al (1989) Nature 342:878-883) or the IMGT numbering system (Lefranc et al, dev. Comparat. Immunol.27:55-77,2003). For a given antibody, one skilled in the art will readily identify the CDRs defined by each numbering system. Also, the correspondence between the different numbering systems is well known to those skilled in the art (see, for example, lefranc et al, dev. Comparat. Immunol.27:55-77,2003).

In the present invention, the CDRs contained in the antibodies or antigen binding fragments thereof of the present invention can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained by an antibody or antigen binding fragment thereof of the invention are preferably determined by Kabat, chothia or IMGT numbering system.

As used herein, the term "framework region" or "FR" residues refer to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

The term "antibody" is not limited by any particular method of producing an antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibodies may be of different isotypes, for example, igG (e.g., igG1, igG2, igG3, or IgG4 subclasses), igA1, igA2, igD, igE, or IgM antibodies.

As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that remains specificThe ability to bind specifically to the same antigen to which the full-length antibody binds, and/or to compete with the full-length antibody for specific binding to an antigen, also referred to as an "antigen-binding portion". See generally Fundamental Immunology, ch.7 (Paul, W., ed., 2 nd edition, raven Press, N.Y. (1989), which is incorporated herein by reference in its entirety for all purposes, antigen binding fragments of antibodies may be generated by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies non-limiting examples of antigen binding fragments include Fab, fab ', F (ab') ₂ Fd, fv, complementarity Determining Region (CDR) fragments, scFv, diabody, single domain antibody (single domain antibody), chimeric antibody, linear antibody (linear antibody), probody and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen binding capacity to the polypeptide. Engineered antibody variants are reviewed in Holliger et al, 2005; nat Biotechnol, 23:1126-1136.

As used herein, the term "full length antibody" means an antibody consisting of two "full length heavy chains" and two "full length light chains". Wherein "full length heavy chain" refers to a polypeptide chain consisting of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a Hinge Region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the N-to C-terminal direction; and, when the full length antibody is an IgE isotype, optionally further comprises a heavy chain constant region CH4 domain. Preferably, a "full length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2 and CH3 in the N-to C-terminal direction. A "full length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-to C-terminal direction. The two pairs of full length antibody chains are linked together by a disulfide bond between CL and CH1 and a disulfide bond between HR of the two full length heavy chains. The full length antibodies of the invention may be from a single species, e.g., human; chimeric or humanized antibodies are also possible. The full length antibodies of the invention comprise two antigen binding sites formed by VH and VL pairs, respectively, which specifically recognize/bind the same antigen.

As used herein, the term "single domain antibody" has the meaning commonly understood by those skilled in the art and refers to an antibody fragment consisting of a single monomer variable antibody domain (e.g., a single heavy chain variable region), typically derived from a variable region of a heavy chain antibody (e.g., a camelid antibody or a shark antibody). Typically, a single domain antibody consists of 4 framework regions and 3 complementarity determining regions, having the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. In certain embodiments, the single domain antibody may be truncated at the N-or C-terminus such that it comprises only a portion of FR1 and/or FR4, or lacks one or both of those framework regions, so long as it substantially retains antigen binding and specificity.

Single domain antibodies, also known as nanobodies, are used interchangeably. In this context, unless the context clearly indicates otherwise, when referring to the term "single domain antibody" it includes not only whole single domain antibodies, but also antigen binding fragments of single domain antibodies.

As used herein, the term "single chain antibody" or "scFv" refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH domains are linked by a linker (linker) (see, e.g., bird et al, science 242:423-426 (1988); huston et al, proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, the Pharmacology of Monoclonal Antibodies, volume 113, roseburg and Moore, springer-Verlag, new York, pages 269-315 (1994)). Such scFv molecules may have the general structure: NH (NH) ₂ -VL-linker-VH-COOH or NH ₂ -VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a polypeptide having an amino acid sequence (GGGGS) can be used ₄ Variants thereof may be used (Holliger et al (1993), proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present invention are described by Alfthan et al (1995), protein Eng.8:725-731, choi et al (2001), eur.J.Immunol.31:94-106, hu et al (1996), cancer Res.56:3055-3061, kipriyanov et al (1999), J.mol.biol.293:41-56 and Roovers et al (2001), cancer Immunol. In some cases, disulfide bonds may also exist between VH and VL of scFv. In certain embodiments of the invention, scFv may form a di-scFv, which refers to two or more singlesThe individual scfvs are serially connected to form antibodies. In certain embodiments of the invention, scFv may be formed (scFv) ₂ It refers to the formation of antibodies from two or more individual scfvs in parallel.

As used herein, the term "Fd" means an antibody fragment consisting of VH and CH1 domains; the term "dAb fragment" means an antibody fragment consisting of a VH domain (Ward et al Nature 341:544 546 (1989)); the term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CH1 domains; the term "F (ab') ₂ Fragment "means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab 'fragment" means a reduction-linked F (ab') ₂ The resulting fragment after disulfide bonding of the two heavy chain fragments in the fragment consists of one complete light and heavy chain Fd fragment (consisting of VH and CH1 domains).

As used herein, the term "Fv" means an antibody fragment consisting of VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antibody fragment that forms the complete antigen binding site. It is believed that the six CDRs confer antigen binding specificity to the antibody. However, even one variable region (e.g., fd fragment, which contains only three CDRs specific for an antigen) is able to recognize and bind antigen, although its affinity may be lower than the complete binding site.

As used herein, the term "Fc" means an antibody fragment formed by disulfide bonding of the second and third constant regions of a first heavy chain of an antibody with the second and third constant regions of a second heavy chain. The Fc fragment of an antibody has a number of different functions, but does not participate in antigen binding.

As used herein, the term "diabody" means that its VH and VL domains are expressed on a single polypeptide chain, but uses a linker that is too short to allow pairing between two domains of the same chain, forcing the domains to pair with complementary domains of the other chain and creating two antigen binding sites (see, e.g., holliger p. Et al, proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak R.J. Et al, structures 2:1121-1123 (1994)).

Antigen-binding fragments of antibodies (e.g., the antibody fragments described above) can be obtained from a given antibody (e.g., an antibody provided by the invention) using conventional techniques known to those of skill in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and specifically screened for antigen-binding fragments in the same manner as used for intact antibodies.

In this context, unless the context clearly indicates otherwise, when referring to the term "antibody" it includes not only whole antibodies, but also antigen-binding fragments of antibodies.

As used herein, the term "chimeric antibody (Chimeric antibody)" refers to an antibody in which a portion of the light chain or/and heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular class or subclass of antibody) and another portion of the light chain or/and heavy chain is derived from another antibody (which may be derived from the same or a different species or belong to the same or a different class or subclass of antibody), but which nevertheless retains binding activity for the antigen of interest (u.s.p 4,816,567 to Cabilly et al.; morrison et al, proc.Natl. Acad.Sci.USA,81:6851 6855 (1984)). In certain embodiments, the term "chimeric antibody" may include antibodies in which the heavy and light chain variable regions of the antibody are from a first antibody and the heavy and light chain constant regions of the antibody are from a second antibody.

As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence is modified to increase homology with the sequence of a human antibody. Typically, all or part of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody) and all or part of the non-CDR regions (e.g., variable region FR and/or constant regions) are derived from a human immunoglobulin (acceptor antibody). In certain embodiments, the CDR regions of the humanized antibody are derived from a non-human antibody (donor antibody) and all or a portion of the non-CDR regions (e.g., the variable region FR and/or constant regions) are derived from a human immunoglobulin (acceptor antibody). Humanized antibodies generally retain the desired properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, and the like. In this application, the donor antibody may be a camelid antibody having a desired property (e.g., antigen specificity, affinity, reactivity, etc.). To prepare humanized antibodies, the CDR regions of an immunized animal can be inserted into a human framework sequence using methods known in the art.

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. The strength or affinity of a specific binding interaction can be determined by the equilibrium dissociation constant (K _D ) And (3) representing. In the present invention, the term "K _D "refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and antigen.

The specific binding properties between two molecules can be determined using methods well known in the art. One method involves measuring the rate of antigen binding site/antigen complex formation and dissociation. Both the "binding rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated from the concentration and the actual rate of association and dissociation (see Malmqvist M, nature,1993, 361:186-187). The kdis/kon ratio is equal to the dissociation constant K _D (see Davies et al, annual Rev Biochem,1990; 59:439-473). K can be measured by any effective method _D Kon and kdis values. In certain embodiments, the dissociation constant may be measured in Biacore using Surface Plasmon Resonance (SPR). In addition to this, bioluminescence interferometry or Kinexa can be used to measure the dissociation constant.

As used herein, a detectable label according to the present invention may be any substance that is detectable by fluorescence, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Such labels are well known in the art, examples of which include, but are not limited to, tag proteins (e.g., flag, his, GST, MBP, HA, myc, etc.), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides The element (e.g., ³ H、 ¹²⁵ I、 ³⁵ S、 ¹⁴ c or ³² P), fluorescent dyes (e.g., fluorescein Isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), texas red, rhodamine, quantum dot or cyanine dye derivatives (e.g., cy7, alexa 750)), luminescent substances (e.g., chemiluminescent substances, such as acridine esters, luminol and derivatives thereof, ruthenium derivatives such as ruthenium terpyridyl), magnetic beads (e.g.,

) A calorimetric label such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to the label-modified avidin (e.g., streptavidin) described above.

As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes, such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuolation virus (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin.

As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as e.g. escherichia coli or bacillus subtilis, a fungal cell such as e.g. yeast cells or aspergillus, an insect cell such as e.g. S2 drosophila cells or Sf9, or an animal cell such as e.g. fibroblasts, CHO cells, COS cells, NSO cells, heLa cells, BHK cells, HEK 293 cells or human cells.

The twenty conventional amino acids referred to herein are written following conventional usage. See, e.g., immunology-a Synthesis (2nd Edition,E.S.Golub and D.R.Gren,Eds, sinauer Associates, sundland, mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally indicated by single-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and active ingredient, which is well known in the art (see, e.g., remington's Pharmaceutical sciences. Mediated by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995), and includes, but is not limited to: pH modifiers, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugar, naCl, and the like. Agents that delay absorption include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline), alcohols and polyols (e.g., glycerol), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. Stabilizers have the meaning commonly understood by those skilled in the art and are capable of stabilizing the desired activity of the active ingredient in a medicament, including but not limited to sodium glutamate, gelatin, SPGA, saccharides (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin or casein) or degradation products thereof (e.g., lactalbumin hydrolysate), and the like. In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solutions (e.g., 0.9% (w/v) NaCl), dextrose solutions (e.g., 5% dextrose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH buffered solutions (e.g., phosphate buffered solutions), ringer's solution, and any combination thereof.

As used herein, the term "preventing" refers to a method that is performed in order to prevent or delay the occurrence of a disease or disorder or symptom in a subject. As used herein, the term "treatment" refers to a method that is performed in order to obtain beneficial or desired clinical results. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., no longer worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and diminishment of symptoms (whether partial or total), whether detectable or undetectable. Furthermore, "treatment" may also refer to an extension of survival compared to the expected survival (if not treated).

As used herein, the term "subject" refers to a mammal, such as a human. In certain embodiments, the subject (e.g., human) has a MSLN positive tumor, or is at risk of having the above-described disease.

As used herein, the term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, the desired effect. For example, an effective amount to prevent a disease (e.g., a tumor involving MSLN positivity) refers to an amount sufficient to prevent, arrest, or delay the onset of the disease; a therapeutically effective amount refers to an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Determination of such effective amounts is well within the ability of those skilled in the art. For example, the amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, and the like.

Advantageous effects of the invention

The chimeric CD3 fusion proteins of the invention and bispecific antibodies (BiTA) against CD3 and mesothelin can activate both edited T cells and unedited T cells without HLA restriction by targeting both MSLN tumor-associated antigen and TCR complex CD3 subunit by the bispecific antibodies. At the same time, the bispecific antibody can activate edited T cells by binding to tumor associated antigens MSLN and chimeric CD3 to release higher levels of bispecific antibody locally in tumors to enhance anti-tumor effects. The invention can realize the organic integration of CAR signal, TCR signal and bispecific antibody activity, to solve the treatment bottleneck of the present cell therapy for solid tumor, to make important breakthrough in the clinical treatment of solid tumor.

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only for illustrating the present invention and are not to be construed as limiting the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the following detailed description of the preferred embodiments and the accompanying drawings.

Drawings

FIG. 1 shows the results of detection of intermediate levels of skin expression in different tumor cell lines.

FIG. 2 shows the structural design of MSLN CAB and its control group (MSLN-BiTA and MSLN-41BB-CD3z (MSLN-CAR)).

FIG. 3 shows the results of detection of the positive rate of MSLN CAB and its control structure engineered T cells (MSLN CAB-T, MSLN BiTA-T and MSLN CAR-T).

FIG. 4 shows the results of detection of the release levels of MSLN CAB-T, MSLN BiTA-T and MSLN CAR-T cytokines IFN-gamma.

FIG. 5 shows the results of detection of the expression of MSLN CAB-T, MSLN BiTA-T, and MSLN CAR-T cell activation markers CD137 (FIG. 5A) and CD69 (FIG. 5B).

FIG. 6 shows the results of detection of CD107a expression levels after activation of MSLN CAB-T, MSLN BiTA-T and MSLN CAR-T cells.

FIG. 7 shows the results of in vitro killing activity assays for MSLN CAB-T, MSLN BiTA-T and MSLN CAR-T cells.

FIG. 8 shows the results of detection of inhibition of MSLN CAB-T cytokines by dasatinib.

FIG. 9 shows the anti-tumor activity of MSLN CAB-T and MSLN CAR-T in immunodeficient mice (NSG).

FIG. 10 shows the effect of MSLN CAB-T and MSLN CAR-T on the body weight of NCI-H596 tumor-bearing immunodeficient mice (NSG).

FIG. 11 shows the in vitro activity advantage of MSLN CAB-T in competing products with the same target. FIG. 11A shows the results of the detection of the positive rates of MSLN CAB-T, BMK1 and BMK 2; FIG. 11B shows the results of detection of the expression of MSLN CAB-T, BMK1 and BMK2 killing activity marker CD107a and cell activation markers CD137 and CD 69; FIG. 11C shows the results of detection of IFN-gamma and IL-2 secretion levels by MSLN CAB-T, BMK1 and BMK2 cytokines; FIG. 11D shows the results of in vitro killing activity of MSLN CAB-T, BMK1 and BMK 2.

Sequence information

A description of the sequences to which the present application relates is provided in the following table.

Table 1: sequence information

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Detailed Description

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.

Unless otherwise indicated, molecular biology experimental methods and immunoassays used in the present invention are basically described in j.sambrook et al, molecular cloning: laboratory Manual, 2 nd edition, cold spring harbor laboratory Press, 1989, and F.M. Ausubel et al, fine-compiled guidelines for molecular biology experiments, 3 rd edition, john Wiley & Sons, inc., 1995; the use of restriction enzymes was in accordance with the conditions recommended by the manufacturer of the product. Those skilled in the art will appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed.

Example 1: expression level of MSLN in different human tumor cells

To identify the expression level of MLSN antigen in different tumor cells, we used flow cytometry analysis to examine mesothelin expression levels of human embryonic kidney cell line HEK 293T, human colorectal adenocarcinoma cell line HT-29, MSLN HT-29 stably transformed cell line (HT-29-MLSN), human lung adenocarcinoma cell line NCI-H596, and ovarian carcinoma cell line OVCAR 3. The specific detection method comprises the following steps:

1) Taking 1×10 pieces respectively ⁵ The cells were placed in 96-well U-bottom plates, 4 wells were placed for each cell, centrifuged at 300g for 5min, and then centrifuged at 200. Mu.L FACS buffer,300g for 5 min;

2) The supernatant after centrifugation was decanted, and 200. Mu.L of FACS buffer was added to resuspend the cells, and the cells were centrifuged at 300g for 5 min;

3) After the supernatant after centrifugation was decanted, each of the above cells was incubated with the following diluted antibodies at 4℃for 30min (100. Mu.L/well) in the absence of light, respectively; antibody dilution was as follows: human Mesothelin PE-conjugated Antibody (R & D, FAB 32652P), FACS buffer 1:500 dilution; anti-MSLN VHH (SEQ ID NO: 1), FACS buffer was diluted to 100nM. For the Mesothelin direct standard antibody staining method, empty cells were used as a control; for anti-MSLN VHH indirect staining, flow-type secondary antibody single staining was used as a control.

4) After co-incubation of the samples, 300g,5min centrifugation, and decanting the supernatant;

5) Add 200. Mu.L FACS buffer to resuspend cells, 300g,5min centrifuge;

6) After repeating step 5 twice, the cell samples stained with Human Mesothelin PE-conjugated Antibody are resuspended in 200 μl FACS buffer and then detected by flow cytometry;

7) After repeating step 5 twice, the cell samples incubated with Anti-MSLN VHH (SEQ ID NO: 1) were incubated for 30min (100. Mu.L/well) with the following diluted fluorescent secondary antibodies, goat F (ab') 2Anti-Human IgG-Fc (PE) (Abcam, cat#ab 98596), respectively; antibody dilution was as follows: the fluorescent secondary antibody, goat F (ab') 2Anti-Human IgG-Fc (PE) (Abcam, cat#ab 98596) was diluted 1:200 with FACS buffer.

8) The diluted fluorescent secondary antibodies were added to the corresponding cells, respectively, and incubated at 4℃for 30min (100. Mu.L/well) in the absence of light. After co-incubation of the samples, 300g,5min centrifugation, and decanting the supernatant;

9) Add 200. Mu.L FACS buffer to resuspend cells, 300g,5min centrifuge;

10 After repeating step 9 twice, the sample stained with the Goat F (ab') 2Anti-Human IgG-Fc (PE) fluorescent secondary antibody is placed in a flow cytometer for detection.

The results of the assay are shown in FIG. 1 and demonstrate the use of commercial antibodies Human Mesothelin PE-conjugated Antibody from R & D, cat: FAB 32652P) can detect MSLN expression in HT-29-MSLN stable transformants, as well as NCI-H896 and OVCAR3, but not in HEK 293T cells and HT-29 cells; the MSLN-targeted nanobody anti-MSLN VHH (SEQ ID NO: 1) provided by the application can be used for detecting the expression of MSLN in HT-29-MSLN stable transgenic lines, NCI-H896 and OVCAR3, and can be used for detecting the micro-expression of MSLN in HT-29 cells, but the expression level of MSLN detected in HEK 293T cells is not obvious.

Example 2: MSLN CAB and design of contrast structure thereof

As previously described, CAB-T is a technique for editing T cells using Chimeric CD3e and a CD3 antibody-based bispecific T cell activator (Chimeric CD3e and anti-CD3 BiTA engineered T cells, CAB-T). The chimeric CD3e (CD 3e-BBζ) in the CAB structure comprises 4 modules of CD3e extracellular domain, CD8 hinge and transmembrane domains, 4-1BB intracellular domain and CD3 ζ intracellular domain; the BiTA structure in CAB structure comprises a single domain antibody sequence (VHH) or single chain antibody variable region sequence (scFv) that recognizes a tumor antigen.

In the invention, in order to verify the anti-tumor activity of MSLN CAB-T, a CAB and a control group structure thereof are designed by utilizing a nano antibody targeting MSLN, and the method comprises the following steps: three structures, MSLN-CAB, MSLN-BiTA and MSLN-41BB-CD3z (MSLN-CAR). MLSN-CAB consists of two structures of cis-expressed chimeric CD3e and MSLN-targeted BiTA; the MLSN-BiTA consists of a fourth extracellular region of the homeopathic ERBB2, a transmembrane region (tERBB 2) and a BiTA structure of a targeting MSLN; MSLN-CAR is a classical MSLN-targeted second generation CAR-T with 4-1BB as co-stimulatory signal. All three structures were provided with FLAG tags for positive rate detection of their edited cells. The specific structure is shown in fig. 2.

Example 3: packaging of lentiviral vectors with MSLN CAB and control constructs thereof

In the invention, MSLN CAB-T and its control group edited T cells are prepared by using lentivirus as a vector. First, the nucleotide sequences shown in SEQ ID NOS.22-24 were introduced into lentiviral vectors, respectively, to prepare lentiviral vectors carrying CAB and a gene encoding a control structure thereof. The specific flow of lentivirus package is as follows:

1) 1X 10 plates were plated in 10cm plates ⁷ HEK 293T cells, 10mL of DMEM (from Hyclone, Cargo number: SH 30243.01) medium, thoroughly mixing the cells, and culturing overnight at 37 degrees;

2) Day 2, to HEK293T (purchased from ATCC, cat: CRL-3216) the cell fusion degree reaches about 90 percent, serum-free DMEM is replaced;

3) Plasmid complexes were prepared, the amounts of each plasmid being: 8 μg of plasma DNA (which contains a nucleotide sequence encoding the MSLN-CAB, MSLN-BiTA or MSLN-41BB-CD3z (MSLN-CAR) structure as described in example 2), 4 μg of psPAX2 and 2 μg of pMD2g, dissolved in 1mL of opti-MEM (available from Gibco, cat# 31985-070) and 42 μl of PEI (available from Polysciences, cat# 24765-2) were added; vortex for 20s. After 15 minutes of standing at room temperature, the mixture was gently added to HEK293T medium along the sides and the culture continued at 37 ℃;

4) After 4h incubation, the medium was removed, PBS (purchased from Hyclone, cat: SH 30256.01) was washed once and fresh DMEM medium pre-warmed with 2% fbs was re-added;

5) Supernatants were collected 48h and 72h after transfection, respectively, and after centrifugation at 2000g for 5 min, the pellet was discarded, and the supernatant was filtered through a 0.25 μm filter (from Sartorius, cat: 16541-K) was filtered and 5% PEG8000 (from Sigma, cat: 89510-1 KG-F) and NaCl at a final concentration of 0.15M (from Sigma, cat: S5150-1L) is mixed vigorously and then is left at 4 ℃ overnight;

6) The virus supernatant was centrifuged at 2000g at 4℃for 20 min, the supernatant removed and the virus pellet was dissolved in 50-100. Mu.L PBS and frozen at-80 ℃.

EXAMPLE 4 preparation of MSLN CAB and control Structure-engineered T cells

After preparation of the lentiviral vector carrying the MSLN-CAB or the control group structure thereof is completed, the lentiviral vector can be used for infecting immune cells to complete preparation of CAB-T cells. The specific procedure for the preparation of CAB-T cells is as follows:

1) Commercial PBMCs (purchased from saichiku organisms, cat No.: SLB-HP 050B) cells were treated with X-VIVO 15 (available from LONZA, cat: 04-418Q) with an initial cell density of 1X 10 ⁶ /mL；

2) anti-CD3/CD28 Beads (purchased from Miltenyi biotec, cat No.: 130-091-441) and 1000IU/mL of IL-2 (purchased from tetracyclic organisms, national drug standard S10970016) was added to activate T cell expansion;

3) After 48 hours of cell activation, the appropriate amount of virus prepared in example 3 and 12 μg/mL protamine (ex Sigma, cat No.: p4005) infected T cells;

4) 24h after lentiviral infection, the cell suspension was aspirated and 1X 10 ⁶ The concentration of cells/mL is supplemented with completely fresh X-VIVO 15 culture medium;

Observing the density of cells every day, and timely supplementing T cell culture solution containing IL-2 1000IU/ml to maintain the density of T cells at 1×10 ⁶ And (5) continuing to amplify the cell/mL for 5-10 days to finish the preparation of the CAR-T cells.

Example 5 detection of MSLN CAB and its control Structure-engineered T cell Positive Rate

After completion of preparation of MSLN CAB-T and its control cells, the infection rate needs to be determined as a basis for subsequent activity analysis. Specifically, the FLAG antibody was used to detect the positive rate of MSLN CAB-T and its control group as follows:

1) Taking 3-5×10 ⁵ Cells were added to each flow tube with 200. Mu.L of FACS buffer (PBS with 1% FBS), 300g,5min centrifuged;

2) 300g, centrifuging for 5min; the centrifuged supernatant was decanted, and 200. Mu.L of 1 XPerm/Wash buffer (from BD bioscience, cat# 554715) was added to resuspend, 400g, and centrifuged for 5min; washing twice;

3) The centrifuged supernatant was decanted and 100 μl of 1:1000FACS buffer diluted Anti-dykdddk antibody (available from BD bioscience, cat: 637310 Mixing the cells uniformly, and incubating for 30min at 4 ℃;

4) After incubation, 1mL of 1 XPerm/Wash buffer,400g,5min centrifugation was added to each flow tube;

5) Pouring out the supernatant after centrifugation, adding 1mL of 1 XPerm/Wash buffer for resuspension, and centrifuging for 400g and 5min;

6) The supernatant after centrifugation was decanted, and 100. Mu.L of 1 XPerm/Wash buffer was added to each sample; the sample was placed in a flow cytometer for detection.

The detection results are shown in figure 3, and the results show that the positive rate of MSLN CAB-T, MSLN BiTA-T and MSLN CAR-T edited T cells is between 22 and 28 percent, the infection rate difference between different samples is within an acceptable range, and the requirements of the next analysis are met.

Example 6 detection of MSLN CAB and its control Structure-engineered T-cell cytokine IFNγ -Release level

When MSLN CAB-T cells are co-cultured with tumor cells, MSLN CAB-T can recognize target antigens on the surface of tumor cells and activate, releasing a large amount of inflammatory cytokines. Based on this, the level of cytokine release by activated MSLN CAB-T cells and their control group was examined using enzyme-linked immunosorbent assay (ELISA) in this example.

The ELISA detection procedure was as follows:

1) Effector cells and target cells each 1×10 ⁵ 200. Mu.L/well. Co-cultured overnight 96-well cell culture plates, 300g,5min centrifuged, 150. Mu.L supernatant/well transferred to new 96-well cell culture plates with a multichannel pipette and cytokine detection was performed using IFN-gamma (ex Invitrogen, cat# 88-7316-88) detection kit, respectively;

2) The ELISA plate was coated with the Human IFN-gamma mAb one day in advance. Diluting the Human IFN-gamma mAb with PBS (1:250), adding 100 mu L of antibody into each well, sealing the ELISA plate with a sealing plate film, and standing at 4 ℃ overnight;

3) Washing the plate: pouring out the liquid in the plate rapidly, adding Wash Buffer with a multi-channel liquid dispenser, and repeatedly washing the plate for five times with 200 mu L of each hole;

4) 200. Mu.L of 1 XELISA/ELISASPOT diluent was added to each well, covered with a sealing plate membrane and blocked at room temperature for 60 minutes;

5) Washing the plate: pouring out the liquid in the plate rapidly, adding Wash Buffer with a multi-channel liquid dispenser, and repeatedly washing the plate for five times with 200 mu L of each hole;

6) Preparation of the Human IFN-. Gamma.ELISA Standard, 8 gradients (units pg/mL) were set: 1000 500, 250, 125, 62.5, 31.25, 15.625,7.8125;

7) Adding standard substances and samples into an ELISA plate, diluting the standard substances and the samples to required concentration by using 1 XELISA/ELISASPOT diluent at 100 mu L/hole, covering a sealing plate film, and incubating for 2 hours at room temperature;

8) Washing the plate: pouring out the liquid in the plate rapidly, adding Wash Buffer with a multi-channel pipettor, and washing the plate four times repeatedly with 200 mu L of liquid in each hole;

9) Diluting the Human IFN-gamma detection antibody with PBS (1:250), adding 100 mu L of antibody into each hole, and sealing the ELISA plate with a sealing plate film for incubation for 1h at room temperature;

10 HRP-conjugated Streptavidin was prepared, diluted with PBS (1:250), and 100. Mu.L per well was added to the ELISA plate. Covering a sealing plate film, and incubating for 30 minutes at room temperature;

11 Plate washing: pouring out the liquid in the plate rapidly, adding Wash Buffer with a multi-channel liquid dispenser, and repeatedly washing the plate for five times with 200 mu L of each hole;

12 30min in advance to restore TMB Substrate to room temperature, 100. Mu.L per well to the ELISA plate. After 5-10 minutes of reaction at room temperature, 50. Mu.L/well Stop solution was added;

13 The absorbance is read by the enzyme label instrument at the detection wavelength od=450 nm;

14 Calculating a standard curve according to the concentration and the OD value of the standard substance, and calculating the concentration of the sample to be detected according to the standard curve. GraphPad Prism mapping software.

The results of the assay are shown in FIG. 4, where MSLN CAB-T, MSLN BiTA-T and MSLN CAR-T can activate and release IFN-gamma cytokines after co-culture with MSLN positive tumor cells HT-29-MSLN, NCI-H596 and OVCAR3, while there is no significant cytokine release after co-culture with MSLN negative HEK 293T and HT-29 cells. Activation of MSLN CAB-T was demonstrated to be target cell MSLN antigen expression dependent.

Example 7 detection of MSLN CAB and its control Structure-engineered T cell activation markers

When MSLN CAB-T cells are co-cultured with tumor cells, MSLN CAB-T can recognize target antigens on the surface of tumor cells and activate, and the expression level of the T cell activation marker proteins including CD137, CD69 and the like on the surface of the membrane can be significantly up-regulated. Based on this, the change in the expression level of the membrane surface protein in activated MSLN CAB-T cells was detected using the staining method and flow cytometry described above in this example.

The specific cell staining procedure is as follows:

1) Effector cells and target cells each 1×10 ⁵ 200. Mu.L/well. Co-cultured overnight 96 well cell culture plates, 300g,5min centrifuged, 200. Mu.L FACS buffer per well, 300g,5min centrifuged;

3) Antibody dilution with FACS buffer (100 μl/well):

4) BV421 Mouse Anti-Human CD3 (available from BD Bioscience, cat: 562426 1:500 dilution

5) PE Mouse Anti-Human CD137 (available from BD Bioscience, cat: 555956 1:200 dilution

6) APC Mouse Anti-Human CD69 (available from BD Bioscience, cat: 555533 1:200 dilution

7) Pouring out the supernatant after centrifugation, adding 100 mu L of antibody mix into each hole, and incubating for 30min at 4 ℃ in a dark place;

8) 200. Mu.L of FACS buffer was added to each well, centrifuged at 300g for 5min, and the supernatant was decanted;

9) After repeating step 5 twice, analyzing the positive cell proportion on a flow cytometer;

10 Flow cytometry, gating on FSC, SSC to obtain desired lymphocyte Populations (PBMCs), selecting CD3BBV421+ therefrom, and analyzing CD137 PE in the CD3 positive cell populations ⁺ Cell and CD69 APC ⁺ Percentage of cells.

As shown in FIG. 5, the expression levels of CD137 and CD69 were significantly up-regulated after co-culturing with MSLN positive tumor cells HT-29-MSLN, NCI-H596 and OVCAR3, and were weakly up-regulated after co-culturing with HT-29, but not with MSLN negative HEK 293T after co-culturing with MSLN CAB-T, MSLN BiTA-T and MSLN CAR-T, respectively. Based on the results in example 1, we believe that HT-29 itself also has some MSLN expression, resulting in an up-regulation of the expression levels of CD137 and CD69 after co-cultivation of MSLN CAB-T or MSLN BiTA-T with HT-29.

Furthermore, we also noted that the proportion of activated cells in the MSLN CAB-T to MSLN BiTA-T sample group was much higher than that in the MSLN CAR-T sample group, indicating that the MSLN BiTA antibodies secreted by MSLN CAB-T to MSLN BiTA-T also successfully activated non-edited T cells in the co-culture system, demonstrating the advantages of MSLN CAB-T to MSLN BiTA-T in terms of anti-tumor.

Example 8 detection of MSLN CAB and its control Structure-engineered T cell activation markers

After co-culturing MSLN CAB-T cells with tumor cells, the killing ability of T cells represented by CD107a was also enhanced in addition to the CD137 and CD69 cell activation markers. Based on this, the change in the expression level of CD107a membrane surface protein in activated MSLN CAB-T cells was detected using flow cytometry in this example.

The specific cell staining procedure is as follows:

1) The MSLN CAB-T cells and the control group cells thereof are resuspended in serum-free medium to adjust the cell concentration to 1X 10 ⁶ Per ml, the fluorescent dye CellTrace Violet (from invitrogen, cat# C34557) was added to a final concentration of 5. Mu.M. Mixing well and incubating at 37 ℃ for 30min.

2) Resuspension the engineered T cells 5-fold volume at room temperature, centrifugation at 1500rpm for 5min, discarding supernatant, and resuspension the cells in X-VIVO medium;

3) Counting to determine the number of cells used, wherein the engineered T cells are 1X 10 ⁵ Well, target cell 2X 10 ⁵ /well. Taking required numbers of engineering T cells and target cells, adding corresponding cells into a 96-well cell culture plate, wherein the total volume of each well is 200uL;

4) 5mL PE Mouse Anti-Human CD107a (available from Biolegend, cat: 328620 At 37℃with 5% CO ₂ Culturing for 1h;

5) 14mL of 1% Golgi stop (from BD, cat: 554724 (i.e., golgiStop was 100-fold diluted with T cell medium) at 37 ℃,5% CO ₂ Culturing for 3.5h;

6) 300g, centrifuging for 5min, pouring out the supernatant after centrifugation, adding 200uL FACS Buffer,300g into each hole, and centrifuging for 5min;

7) Pouring out the supernatant after centrifugation, and repeating the operation 6;

8) The cells were resuspended and homogenized with 100ul FACS buffer

9) Flow cytometry detects the percentage of CD107a pe+ cells in CTV positive T cells.

As shown in FIG. 6, the expression levels of CD107a were significantly up-regulated after co-culturing with MSLN positive tumor cells HT-29-MSLN, NCI-H596 and OVCAR3, and slightly up-regulated after co-culturing with HT-29, whereas MSLN CAB-T, MSLN BiTA-T and MSLN CAR-T were not significantly up-regulated after co-culturing with MSLN negative HEK 293T, respectively. The results are consistent with those of example 7.

Example 9 detection of MSLN CAB and control structure engineered T cell in vitro killing activity of MSLN CAB-T cells whether or not MSLN CAB-T cells have in vitro killing activity is a key basis for determining the potential clinical efficacy of MSLN CAB-T. To verify tumor killing activity of MSLN CAB-T, we tested using LDH method.

The specific experimental procedure is as follows:

1) Respectively setting an experimental hole, an effector cell control hole, a target cell maximum release hole, a culture medium control hole and a volume control hole; experimental procedure according to Cytotox

Standard procedure of Non-Radioactive Cytotoxicity Assay kit (from Promega, cat# G1781);

2) Setting different effective target ratios, namely effector cell number: target cell number = 0:1,1:1,5:1,10: 1,20: 1, a step of;

cell number: target cells 1X 10 ⁴ 50. Mu.L/well;

experimental wells, effector cells with different dilution ratios were added: target cell = 0:1,1:1,5:1,10: 1,20: 1 (effector cells 50. Mu.L+target cells 50. Mu.L) to a cell culture plate, 3 replicates were set;

effector cell control wells, i.e., effector cells: target cell = 0:0,1:0,5:0,10: 0,20: 0, setting 2 repeats;

target cell control well, 1X 10 target cells were added ⁴ Well, 50 μl+50 μl of medium;

maximum release of target cells, i.e.1X 10 addition of target cells ⁴ 50. Mu.L+50. Mu.L of medium, 10. Mu.L of lysate was added 1h before collection;

culture medium control wells, i.e., 100 μl of culture medium was added;

volume control wells, i.e., 100. Mu.L of medium was added, 10. Mu.L of lysate was added to the wells with maximum release of target cells 1h before collection, and incubated at 37 ℃.

3) According to the designed plate layout, sample adding, placing a plate for laying at 37 ℃, and incubating for 24 hours, 36 hours or 48 hours by 5% CO 2;

4) The detection buffer (assay buffer) was taken out from the-20℃refrigerator and dissolved in the 4℃refrigerator, taking care of light protection. In application, 12ml of detection buffer (assay buffer) is added to a bottle bottom mixture (substrate Mix) and mixed well.

5) Placing the culture plate in 250g for 4min for centrifugation, and transferring 50 mu L/hole cell supernatant to a new enzyme-labeled plate;

6) Adding 50 mu L/hole substrate mixture (12 mL assay buffer to a bottle of substrate Mix to Mix well) to the new enzyme-labeled plate;

7) Incubating for 30min at room temperature in dark place, and adding 50 mu L/Kong Zhongzhi solution;

8) The absorbance was read by the microplate reader at detection wavelength od=490 nm, completed within 1 h. Based on the OD values, the cell killing ratio (percent%) was calculated:

Experimental well = effective target ratio-medium control (mean value)

Target cell spontaneous = target cell control-medium control (mean value)

Effector spontaneous = effector control-culture medium control (mean)

Maximum release of target cells = maximum release of target cells (mean) -volume control (mean)

Cell killing ratio (%) = (experiment-target cell spontaneous-effector cell spontaneous)/(target cell maximum release-target cell spontaneous)

As shown in FIG. 7, the killing activity of MSLN CAB-T on MSLN positive NCI-H596 and OVCAR3 cells was superior to that of MSLN BiTA-T and MSLN CAR-T, and MSLN CAB-T on MSLN weak positive HT-29 cells showed some killing at a high ratio of effector to target cells, but no killing activity on MSLN negative HEK 293T cells.

Example 10 dasatinib was effective in inhibiting cytokine release from MSLN CAB-T

Immune cell therapy, which is an active cell drug, rapidly releases cytokines into the blood after encountering antigen stimulation, resulting in dangerous hyperthermia, rapid drop of blood pressure, and other symptoms in patients. Therefore, to improve the controllability and safety of MSLN CAB-T cells, we chose to inhibit MSLN CAB-T cell activity in vitro with Dasatinib (Dasatinib) as a safety switch. Based on this, the inhibition of MSLN CAB-T cytokine level by dasatinib was examined by enzyme-linked immunosorbent assay (ELISA) in this example.

The ELISA detection procedure was as follows:

1) Effector cells and target cells each 1×10 ⁵ 200. Mu.L/well. 100nM,50nM,25nM,12.5nM,6.25nM,3.125nM,0nM of dasatinib (available from sigma, cat. No. CDS023389-25 MG) were added simultaneously. Co-cultured overnight 96-well cell culture plates, 300g,5min centrifuged, 150. Mu.L of supernatant/well transferred to new 96-well cell culture plates with a multichannel pipette and cytokine detection was performed using IFN-gamma (ex Invitrogen, cat# 88-7316-88)/IL-2 (ex Invitrogen, cat# 88-7025-88) detection kit, respectively;

2) The ELISA plate was coated with Human IFN-gamma/IL-2/mAb one day in advance. Diluting the Human IFN-gamma/IL-2 mAb with PBS (1:250), adding 100 mu L of antibody into each well, sealing the ELISA plate with a sealing plate film, and standing at 4 ℃ overnight;

9) Diluting the Human IFN-gamma/IL-2 detection antibody with PBS (1:250), adding 100 mu L of antibody into each hole, and sealing an ELISA plate by using a sealing plate film for incubation for 1h at room temperature;

13 The absorbance is read by the enzyme label instrument at the detection wavelength od=450 nm; and calculating a standard curve according to the concentration and the OD value of the standard substance, and calculating the concentration of the sample to be detected according to the standard curve. GraphPad Prism mapping software.

As shown in FIG. 8, 3.125nM dasatinib was effective in inhibiting the release of cytokines IL-2 and IFN-gamma from MSLN CAB-T co-cultured with MSLN positive tumor cells NCI-H596. Implication, dasatinib would have potential to be used clinically as a safety switch to control MSLN CAB-T activity.

EXAMPLE 11 MSLN CAB and its control Structure-engineered T-cell tumor growth inhibition in humanized mouse tumor model

Tumor inhibition of MSLN CAB-T cells in a mouse tumor model is a key basis for judging whether MSLN CAB-T has clinical efficacy in the future. To verify the anti-tumor activity of MSLN CAB-T in mice, we examined the anti-tumor activity of MSLN CAB-T in NSG immunodeficient mouse model vaccinated with human lung adenocarcinoma NCI-H596 tumor cells.

The specific experimental procedure is as follows:

NCI-H596 cell expansion culture, inoculation:

1) In vitro culture to expand enough NCI-H596 cells, pancreatin digestion and cell collection, PBS washing 3 times, counting, 80% RPMI-1640 basal medium plus 20% Matrigel to adjust cell density to 20×10 ⁶ Packing cells/ml into a 50ml centrifuge tube, covering the centrifuge tube orifice tightly, sealing by a sealing film, and introducing into an SPF-level animal house through a transmission window;

2) After adaptively feeding pre-purchased 33 female NSG severe immunodeficiency mice of 8 weeks of age for 1 week, the right abdominal mouse hair of each mouse was removed with a shaver. The density is 20 multiplied by 10 ⁶ The cells/ml NCI-H596 cells were thoroughly mixed by pipetting with 1ml pipettor, and the cells were inoculated subcutaneously into the right flank of each NSG mouse with 1ml syringe and 0.2ml each mouse was inoculated, i.e., 4X 10 each NSG mouse was inoculated ⁶ The cells were observed daily for subcutaneous neoplasia in NSG mice, and each NSG mouse was numbered 20 days after inoculation using an ear tag with a number;

NCI-H596 tumor bearing mice were grouped, dosed, measured:

1) After 20 days of inoculation, the maximum axis W and maximum axis L of each NSG right-hand abdominal subcutaneous tumor were measured using vernier calipers, and the body weight of each mouse was weighed using an electronic balance. The right flank subcutaneous tumor volume was calculated for each NSG mouse as tumor volume t=1/2×w×w×l. Rejecting mice with oversized tumor volume and undersized tumor volume, and equally dividing NSG mice into 5 groups of 6 mice according to average tumor volume;

2) Grouping and injecting the corresponding reagents or cells according to the group dosing regimen shown in Table 2

Table 2 group dosing regimen

The tumor growth curves of the mice in each group are shown in fig. 9, and the tumors of the mice in the PBS group and the NT group are gradually increased along with the extension of the inoculation time, which indicates that the CDX transplantation model of the experiment is successfully established; compared with the PBS group and the NT group, the MSLN CAR-T cell treatment group shows the inhibition effect of medium level tumor growth, and the MSLN-CAB-T cell treatment group shows the tumor growth inhibition effect superior to that of the MSLN CAR-T. Throughout the experimental period, there was no significant drop in body weight for each group of mice (fig. 10), indicating good safety for each test sample.

Example 12 in vitro Activity advantage of MSLN CAB-T over off-target bid

To further analyze the in vitro activity advantage of MSLN CAB-T cells against commercial co-target competing products, we selected the MSLN targeting CAR sequences disclosed in the U.S. Pat. No. 2020/0407460 A1, and TCR, filed by the university of Nohua and pennsylvania, incorporated herein by reference ² MSLN targeting TFR (TCR receptor fusion protein) sequences disclosed in the patent application of Therapeutics, inc. (US 2021/0079057 A1) and the corresponding sequences were constructed separately into lentiviral vectors for the preparation of corresponding edited T cells designated BMK1 (MSLN CAR-T) and BMK2 (MSLN TFR-T), respectively.

For lentiviral packaging methods and methods for preparing edited T cells please refer to example 3 and example 4, respectively. The positive rate results of the edited T cells are shown in FIG. 11A, and the positive rates of MSLN CAB-T, MSLN CAR-T (BMK 1) and MSLN TFR-T (BMK 2) are respectively: 46.45%,41.25% and 39.10%. The positive rate meets the requirement of activity contrast analysis.

The methods for analysis of the MSLN CAB-T and its bid T cell activation markers CD69 and CD137, and the in vitro killing activity marker CD107a are referred to example 7 and example 8, respectively. As shown in fig. 11B, after co-culturing MSLN CAB-T and its bid products BMK1 and BMK2 with MSLN positive tumor cells MSLN-HT29, NCI-H596 and OVCAR3, respectively, for 24 hours, MSLN CAB-T expressed killing activity marker CD107a has significant advantages over both BMK1 and BMK 2; furthermore, the T cell activation markers CD69 and CD137 expressed by MSLN CAB-T also exhibited expression levels comparable to or better than BMK1 and BMK 2.

The detection method of cytokines IFN-gamma and IL-2 released after activation of MSLN CAB-T and its bid is described in examples 6 and 10. Specifically, MSLN CAB-T and its bid products BMK1 and BMK2 were co-cultured with MSLN positive tumor cells MSLN-HT29, NCI-H596 and OVCAR3, respectively, for 24 hours, and supernatants were collected and assayed for secretion levels of cytokines IFN-gamma and IL-2, respectively. As a result, as shown in FIG. 11C, MSLN CAB-T secreted IFN- γ at a level substantially equivalent to BMK1, but much greater than that of BMK 2; in terms of IL-2 secretion, MSLN CAB-T is comparable to or higher than BMK2 but lower than or comparable to BMK 1.

Comparative experiments on the in vitro killing activity of MSLN CAB-T and the bid product are described in example 9. Specifically, MSLN CAB-T and its bid products BMK1 and BMK2 were co-cultured with MSLN negative HEK293T cells or MSLN positive tumor cells MSLN-HT29, NCI-H596 and OVCAR3, respectively, for 24 hours, and then the content of LDH in the supernatant was examined. From the results of FIG. 11D, MSLN CAB-T has no killing activity against MSLN negative cells, while killing of MLSN positive tumor cells is superior to, or substantially equivalent to, BMK1 and BMK 2.

Combining the detection results of T cell activation markers, killing markers, cytokine secretion levels and killing tests, MSLN CAB-T has certain advantages compared with BMK1 and BMK 2. It is believed that the advantage observed in the experiments was mainly superior to MSLN CAB-T mobilizing the antitumor activity of unedited T cells in the culture system.

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate that: many modifications and variations of details may be made to adapt to a particular situation and the invention is intended to be within the scope of the invention. The full scope of the invention is given by the appended claims together with any equivalents thereof.

Claims

1. A bispecific antibody construct comprising a first binding domain that specifically binds MSLN and a second binding domain that specifically binds CD 3; wherein the first binding domain is a single domain antibody or a tandem or oligomeric form thereof, the single domain antibody comprising:

(c) CDR3 consisting of the sequence: SEQ ID NO. 4, or a sequence having a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids) as compared thereto;

Preferably, the first binding domain comprises a CDR1 as shown in SEQ ID NO. 2, a CDR2 as shown in SEQ ID NO. 3, and a CDR3 as shown in SEQ ID NO. 4;

preferably, the first binding domain comprises a sequence as set forth in SEQ ID NO. 1, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) as compared to SEQ ID NO. 1;

preferably, the first binding domain comprises the amino acid sequence shown as SEQ ID NO. 1.

2. The bispecific antibody construct of claim 1, wherein the second binding domain is selected from an antibody or antigen-binding fragment thereof that specifically binds CD 3;

preferably, the antigen binding fragment is selected from a single domain antibody, single chain antibody, fab or tandem or oligomeric forms thereof;

preferably, the second binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) of a monoclonal antibody selected from the group consisting of: L2K, UCHT, OKT, F6A, SP34;

preferably, the second binding domain comprises a VH as shown in SEQ ID No. 5, and/or a VL as shown in SEQ ID No. 6;

preferably, the second binding domain comprises the amino acid sequence shown as SEQ ID NO. 7.

3. The bispecific antibody construct of claim 1 or 2, wherein the first binding domain is linked to the N-terminus and/or C-terminus (e.g. N-terminus) of the second binding domain, optionally via a peptide linker;

preferably, the first binding domain is linked to the N-terminus of the second binding domain by a peptide linker;

preferably, the peptide linker is a peptide linker comprising one or more glycine and/or one or more serine.

4. A bispecific antibody construct according to any one of claims 1 to 3, wherein the bispecific antibody construct comprises the amino acid sequence shown in SEQ ID No. 9.

5. The bispecific antibody construct of any one of claims 1-4, wherein the bispecific antibody construct further comprises a signal peptide and/or a detectable label (e.g. a tag protein);

preferably, the bispecific antibody construct comprises a signal peptide at its N-terminus;

preferably, the bispecific antibody construct comprises a detectable label at its C-terminus.

6. An isolated nucleic acid molecule encoding the bispecific antibody construct of any one of claims 1-5.

7. A vector comprising the isolated nucleic acid molecule of claim 5;

preferably, the vector is a cloning vector or an expression vector; preferably, the vector is a lentiviral, adenoviral or retroviral vector.

8. A host cell comprising the isolated nucleic acid molecule of claim 6 or the vector of claim 7;

preferably, the host cell is an engineered immune cell;

preferably, the engineered immune cell secretes and expresses the bispecific antibody construct of any one of claims 1-5;

preferably, the engineered immune cell is a T cell, NK cell, γδ T cell, NKT cell, or any combination thereof.

9. A composition comprising the bispecific antibody construct of any one of claims 1-5, and a chimeric CD3 fusion protein, wherein the chimeric CD3 fusion protein comprises a polypeptide binding domain comprising a polypeptide that is recognized by a second binding domain of the bispecific antibody construct; optionally one or more of the following domains:

(a) A joint or hinge region;

(b) A transmembrane domain;

(c) Costimulatory signaling domains

(d) A CD3 signaling activation domain;

preferably, the chimeric CD3 fusion protein comprises the polypeptide binding domain and a transmembrane domain from N-terminus to C-terminus; preferably, the chimeric CD3 fusion protein further comprises a costimulatory signaling domain and/or a CD3 signaling activation domain at the C-terminus of the transmembrane domain; preferably, the chimeric CD3 fusion protein further comprises a linker or hinge region between the polypeptide binding domain and the transmembrane domain;

Preferably, the chimeric CD3 fusion protein comprises, in order from the N-terminus to the C-terminus, the polypeptide binding domain, a linker or hinge region, a transmembrane domain, a costimulatory signaling domain, and a CD3 signaling domain.

10. The composition of claim 9, wherein the polypeptide recognizable by the second binding domain comprises or consists of a CD3e extracellular region or fragment thereof;

preferably, the polypeptide recognizable by the second binding domain comprises or consists of amino acids 1-104 of the CD3e protein;

preferably, the polypeptide recognized by the second binding domain comprises the sequence shown in SEQ ID NO. 11.

11. The composition of claim 9 or 10, wherein the hinge region is a hinge region selected from the group consisting of: CD8, CD28, 4-1BB, or any combination thereof;

preferably, the hinge region is a hinge region of CD 8; preferably, the hinge region comprises or consists of the amino acid sequence shown in SEQ ID NO. 12.

12. The composition of any one of claims 9-11, wherein the transmembrane domain is a transmembrane domain selected from the group consisting of: CD8, CD28, CD3, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, 4-1BB, CD154, or any combination thereof;

Preferably, the transmembrane domain is the transmembrane domain of CD 8; preferably, the transmembrane domain comprises or consists of the amino acid sequence shown in SEQ ID NO. 13.

13. The composition of any one of claims 9-12, wherein the costimulatory signaling domain is an intracellular domain selected from the group consisting of: CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134,4-1BB, PD1, DAPLO, CDS, ICAM-1, LFA-1 (CDLLA/CD 18), ICOS (CD 278), NKG2D, GITR, TLR2, or any combination thereof;

preferably, the costimulatory signaling domain is the intracellular domain of 4-1 BB; preferably, the costimulatory signaling domain comprises or consists of the amino acid sequence depicted as SEQ ID NO. 14.

14. The composition of any one of claims 9-13, wherein the CD3 signaling activation domain is a cd3ζ intracellular domain; preferably, the CD3 signal activation domain comprises or consists of the amino acid sequence shown as SEQ ID NO. 15.

15. The composition of any one of claims 9-14, wherein the chimeric CD3 fusion protein comprises an amino acid sequence as set forth in SEQ ID No. 16.

16. The composition of any one of claims 9-15, wherein the chimeric CD3 fusion protein further comprises a signal peptide and/or a detectable label (e.g., a tag protein);

Preferably, the chimeric CD3 fusion protein comprises a signal peptide at its N-terminus;

preferably, the chimeric CD3 fusion protein comprises a detectable label at its C-terminus.

17. An isolated nucleic acid molecule encoding a bispecific antibody construct as defined in any one of claims 1 to 5 and a chimeric CD3 fusion protein as defined in any one of claims 9 to 16;

preferably, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding the bispecific antibody construct and a second nucleotide sequence encoding the chimeric CD3 fusion protein;

preferably, the first nucleotide sequence and the second nucleotide sequence are present on different isolated nucleic acid molecules;

preferably, the first nucleotide sequence and the second nucleotide sequence are present on the same isolated nucleic acid molecule in any order; preferably, the first nucleotide sequence and the second nucleotide sequence are linked in any order by a nucleotide sequence encoding a self-cleaving peptide (e.g. T2A);

preferably, the first nucleotide sequence and/or the second nucleotide sequence optionally further comprises a nucleotide sequence encoding a signal peptide at its 5' end.

18. A vector comprising the isolated nucleic acid molecule of claim 17.

19. The vector of claim 18, comprising a first nucleotide sequence encoding the bispecific antibody construct and a second nucleotide sequence encoding the chimeric CD3 fusion protein, wherein the first nucleotide sequence and the second nucleotide sequence are located in the same expression cassette in any order;

preferably, the first nucleotide sequence and the second nucleotide sequence are linked in any order by a nucleotide sequence encoding a self-cleaving peptide (e.g. T2A);

20. The vector of claim 18, comprising a first nucleotide sequence encoding the bispecific antibody construct and a second nucleotide sequence encoding the chimeric CD3 fusion protein, wherein the first nucleotide sequence and the second nucleotide sequence are located in different expression cassettes;

preferably, the first expression cassette comprising the first nucleotide sequence is located on the same vector or a different vector than the second expression cassette comprising the second nucleotide sequence;

21. An engineered immune cell comprising the isolated nucleic acid molecule of claim 17, or the vector of any one of claims 18-20;

preferably, the engineered immune cell expresses a chimeric CD3 fusion protein as defined in any one of claims 9-16 and a bispecific antibody construct as defined in any one of claims 1-5;

preferably, the engineered immune cell secretly expresses the bispecific antibody construct;

preferably, the engineered immune cell expresses the chimeric CD3 fusion protein on its surface;

22. An immune cell composition comprising the host cell of claim 8 and/or the engineered immune cell of claim 21;

preferably, the immune cell composition further comprises an unedited and/or unsuccessfully edited immune cell;

Preferably, the engineered immune cell count comprises at least 10% of the total number of cells of the immune cell composition.

23. A method of making the host cell of claim 8 or the engineered immune cell of claim 21, comprising: (1) providing an immune cell; (2) Introducing the isolated nucleic acid molecule of claim 6 or 17 or the vector of any one of claims 7, 18-20 into the immune cell;

preferably, the immune cells are selected from T cells, NK cells, γδ T cells, NKT cells, or any combination thereof;

preferably, in step (1), the immune cells are pre-treated, the pre-treatment comprising sorting, activation and/or proliferation of immune cells;

preferably, the nucleic acid molecule or vector is introduced into the host cell in step (2) by means of viral infection or by transfection with a non-viral vector;

preferably, step (2) is followed by a step of amplifying the immune cells obtained in step (2).

24. A composition comprising the bispecific antibody construct of any one of claims 1-5, and an immune cell expressing a chimeric CD3 fusion protein; wherein the chimeric CD3 fusion protein is as defined in any one of claims 9-16;

Preferably, the immune cells are selected from T cells, NK cells, γδ T cells, NKT cells, or any combination thereof.

25. A pharmaceutical composition comprising the bispecific antibody construct of any one of claims 1 to 5, the isolated nucleic acid molecule of claim 6, the vector of claim 7, the host cell of claim 8, the composition of any one of claims 9 to 16, the isolated nucleic acid molecule of claim 17, the vector of any one of claims 18 to 20, the engineered immune cell of claim 21, the immune cell composition of claim 22, or the composition of claim 24;

preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or excipient;

preferably, the pharmaceutical composition comprises the host cell of claim 8, the engineered immune cell of claim 21 or the immune cell composition of claim 22.

26. Use of the bispecific antibody construct of any one of claims 1-5, the isolated nucleic acid molecule of claim 6, the vector of claim 7, the host cell of claim 8, the composition of any one of claims 9-16, the isolated nucleic acid molecule of claim 17, the vector of any one of claims 18-20, the engineered immune cell of claim 21, the immune cell composition of claim 22, the composition of claim 24, or the pharmaceutical composition of claim 25 for the preparation of a medicament for the prevention and/or treatment of a tumor in a subject;

Preferably, the tumor is a MSLN positive tumor;

preferably, the tumor is selected from solid tumors, such as mesothelioma, ovarian cancer, pancreatic cancer, sarcoma, lung adenocarcinoma, endometrial cancer, gastric adenocarcinoma, esophageal cancer, colorectal cancer, breast cancer, small intestine adenocarcinoma, ampulla cancer, uterine sarcoma, parotid adenoma, anal canal cancer, vaginal cancer, throat cancer, head and neck squamous carcinoma, nasal cancer, bile duct cancer or cervical cancer;

preferably, the subject is a mammal, such as a human;

preferably, the bispecific antibody construct, isolated nucleic acid molecule, vector, host cell, composition, isolated nucleic acid molecule, vector, engineered immune cell, immune cell composition, or pharmaceutical composition is used alone or in combination with another pharmaceutically active agent (e.g., an antineoplastic agent);

preferably, the bispecific antibody construct, isolated nucleic acid molecule, vector, host cell, composition, isolated nucleic acid molecule, vector, engineered immune cell, immune cell composition, or pharmaceutical composition is used in combination with dasatinib, e.g., administered simultaneously or sequentially.