CN117126294A

CN117126294A - Signal transduction receptor based on TGFbeta antibody

Info

Publication number: CN117126294A
Application number: CN202310607453.XA
Authority: CN
Inventors: 金华君; 刘天怡; 黄晨
Original assignee: Shanghai Junsai Biotechnology Co ltd
Current assignee: Shanghai Junsai Biotechnology Co ltd
Priority date: 2022-05-27
Filing date: 2023-05-26
Publication date: 2023-11-28

Abstract

The invention relates to a TGF-beta antibody-based signal transduction receptor, in particular to an isolated fusion protein comprising: an anti-TGF-beta antibody or antigen-binding fragment or mutant thereof; a transmembrane region or mutant thereof; and an intracellular domain of a costimulatory signaling molecule, or a functional fragment or mutant thereof. The signal transduction receptor of the present invention can activate intracellular domains more effectively, and further enhance the activation and proliferation levels of immune effector cells expressing the signal transduction receptor.

Description

Signal transduction receptor based on TGFbeta antibody

Technical Field

The invention relates to the field of biotechnology, in particular to a signal conversion receptor and application thereof.

Background

In Adoptive Cell Therapy (ACT) of tumors, stability of the immune system is critical. Too low a reaction may cause severe infection, and too strong a reaction may cause allergic reaction. The human immune system evolved a fine, complex bi-directional immune regulation mechanism that positively and negatively regulated the immune response. Activating and enhancing immune response when foreign antigenic material is removed; after removal of foreign antigenic material, the immune response may be attenuated to termination. In tumor ACT, positive regulatory molecules of effector T cells include CD3/TCR complex, CD28, CD134 (OX 40), CD137 (4-1 BB), etc., and negative regulatory molecules include PD-1 (PDCD 1, CD 279), CTLA-4 (CD 152), LAG3 (CD 223), TIM3 (HAVCR 2), etc. However, in the Tumor Microenvironment (TME), there are many factors that negatively regulate T cell immune responses, allowing tumor cells to escape from the monitoring and clearance of the immune system of the body and continue to proliferate, invade and metastasize.

TGF-beta has an inhibitory effect on the systemic immune system and inhibits immune surveillance of the host. TGF-beta plays an important role in the initiation and progression of tumors. TGF-beta has a tumor promoting effect, which is often produced in large amounts in many types of tumors and is known to have carcinogenesis, and which has a negative impact on tumor immunity and significantly inhibits tumor immunity monitoring of the host. TGF-beta significantly and directly inhibits the cytotoxic process (cytotoxic program) of Cytotoxic T Lymphocytes (CTLs) by inhibiting transcription of genes encoding a number of key proteins, including at least: perforin, granzyme a, granzyme B, fas ligand, and g interferon. Meanwhile, TGF-beta has a significant effect on differentiation and function of CD4+ T cells.

Aiming at the negative regulation inhibitory signal path of immune effector cells, in recent years, a plurality of reports have been made on connecting the extracellular region of the corresponding receptor with the transmembrane region and the intracellular region of a positive co-stimulatory signal molecule, so as to construct a novel fusion receptor, the fusion receptor has the function of a signal converter after being expressed in the immune effector cells, and can convert the immune inhibitory signal in the tumor microenvironment into an immune activation signal, namely, the signal conversion molecule extracellular polypeptide receives the immune inhibitory molecule signal on the surface of the tumor cells, the surface of the tumor stromal cells or the tumor microenvironment and transmits the immune inhibitory molecule signal into cells, and the second signal of the immune cells is activated through the intracellular segment of the co-stimulatory signal molecule, so that the proliferation and cytokine secretion function of the immune effector cells are enhanced, and the survival time of the activated immune effector cells is prolonged. WO2021244486A1 discloses a signaling receptor based on the extracellular region of TGF-beta receptor II, which comprises the extracellular region of TGF-betaRII and transmembrane and intracellular activation domains from other molecules. The TCR-T cells expressing the signal conversion receptor have remarkable promotion on proliferation and in-vitro and in-vivo killing effects on tumor target cells. CN105452287a discloses an immunosuppressive TGF-beta signal transducer which is a chimera comprising an extracellular domain of a TGF-beta receptor and an intracellular domain from another molecule, such that binding of TGF-beta to the extracellular domain results in the intracellular domain stimulating T cell activity.

In order to stimulate proliferation of immune effector cells and enhance their tumor immunity effects, there is still a need for more improved means, including signal transducer molecules, to modify and activate immune effector cells.

Disclosure of Invention

The present invention provides an isolated fusion protein comprising: an anti-TGF-beta antibody or antigen-binding fragment or mutant thereof; a transmembrane region or mutant thereof; and an intracellular domain of a costimulatory signaling molecule or a functional fragment or mutant thereof that retains the costimulatory signaling molecule to deliver a costimulatory signal, activate a biological function of an immune cell.

In one or more embodiments, the anti-TGF-beta antibody is an anti-TGF-beta 1 antibody, an anti-TGF-beta 2 antibody, or an anti-TGF-beta 3 antibody.

In one or more embodiments, the antibody comprises any one or more selected from the group consisting of single chain antibodies (scFv), single domain antibodies (sdAb), nanobodies (nanobodies), heavy chain antibodies (hcabs), fab ', and F (ab') 2.

In one or more embodiments, the antibody has the following HCDR:

the HCDR1 amino acid sequence is shown as SEQ ID NO. 44, the HCDR2 amino acid sequence is shown as SEQ ID NO. 45, and the HCDR3 amino acid sequence is shown as SEQ ID NO. 46.

In one or more embodiments, the antibody has an LCDR as follows:

the amino acid sequence of LCDR1 is shown as SEQ ID NO. 47, the amino acid sequence of LCDR2 is shown as SEQ ID NO. 48, and the amino acid sequence of LCDR3 is shown as SEQ ID NO. 49.

In one or more embodiments, the amino acid sequence of the VH of the antibody is shown as SEQ ID NO. 42.

In one or more embodiments, the amino acid sequence of the VL of the antibody is set forth in SEQ ID NO. 43.

In one or more embodiments, the antibody is a single chain antibody as set forth in SEQ ID NO. 4.

In one or more embodiments, the costimulatory signaling molecules include one or more of CD28, CD134 (OX 40), CD137 (4-1 BB), LCK, ICOS, DAP10, siglec-9, siglec-10, siglec-15, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2R, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21R, CD27, and CD 40. In one or more embodiments, the costimulatory signaling molecules comprise CD28 and/or IL7Ralpha.

In one or more embodiments, the transmembrane region includes any one or more of the transmembrane regions from CD28, CD134 (OX 40), CD137 (4-1 BB), LCK, ICOS, DAP, siglec-9, siglec-10, siglec-15, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2R, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21R, CD27, and CD 40.

In one or more embodiments, the IL-2R, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21R is an alpha, beta, or gamma subunit, respectively.

In one or more embodiments, the transmembrane region is from a CD28 transmembrane region or a mutant thereof or an IL-7Ralpha transmembrane region or a mutant thereof.

In one or more embodiments, the amino acid sequence of the CD28 transmembrane region is shown in SEQ ID NO. 6.

In one or more embodiments, the amino acid sequence of the IL-7Ralpha transmembrane region is shown in SEQ ID NO. 12.

In one or more embodiments, the amino acid sequence of the mutant of the IL-7Ralpha transmembrane region is as shown in any one of SEQ ID NOs 14, 16, 18, 20.

In one or more embodiments, the extracellular region of the fusion protein is a TGF-beta single chain antibody, the transmembrane region is from a CD28 transmembrane region, and the intracellular domain of the costimulatory molecule is the CD28 intracellular domain.

In one or more embodiments, the extracellular region of the fusion protein is a TGF-beta single chain antibody, the transmembrane region is from an IL7Ralpha transmembrane region or a mutant thereof, and the intracellular domain of the costimulatory molecule is any one or more selected from the group consisting of a CD28 intracellular region, an IL7Ralpha intracellular region, a CD134 intracellular region, a CD137 intracellular region, an IL-2Rbeta intracellular region, an IL-4Rbeta intracellular region, an IL-7Ralpha intracellular region, an IL-10Ralpha intracellular region, an IL-12Rbeta intracellular region, an IL-15Ralpha intracellular region, an IL-21Ralpha intracellular region, a CD27 intracellular region, and a CD40 intracellular region.

In one or more embodiments, the amino acid sequence of the CD28 intracellular region is shown in SEQ ID NO. 8.

In one or more embodiments, the amino acid sequence of the CD134 intracellular region is shown in SEQ ID NO. 51.

In one or more embodiments, the amino acid sequence of the IL-7Rα intracellular region is shown in SEQ ID NO. 10.

In one or more embodiments, the signal transduction receptor molecule further comprises a membrane surface tag comprising a BCMA extracellular domain or variant thereof.

In one or more embodiments, the membrane surface tag further comprises a linker or hinge region located at the N-terminus or C-terminus of the BCMA extracellular domain or variant thereof.

In one or more embodiments, the linker has an amino acid sequence as set forth in SEQ ID NO. 24 or 26.

The invention also provides a polynucleotide molecule selected from the group consisting of: a polynucleotide molecule or complementary sequence encoding a fusion protein according to any one of the embodiments of the invention.

In one or more embodiments, the fusion protein has an anti-TGF-beta antibody encoding sequence as set forth in SEQ ID NO. 3.

In one or more embodiments, the transmembrane region of the fusion protein is a CD28 transmembrane region having the coding sequence shown in SEQ ID NO. 5.

In one or more embodiments, the transmembrane region of the fusion protein is an IL7Ralpha transmembrane region having a coding sequence as set forth in SEQ ID NO. 11.

In one or more embodiments, the intracellular domain of the costimulatory signaling molecule in the fusion protein is the CD28 intracellular region, the coding sequence of which is shown in SEQ ID NO. 7.

In one or more embodiments, the intracellular domain of the costimulatory signaling molecule comprises a CD28 intracellular region having the coding sequence shown in SEQ ID NO. 7 and a CD134 intracellular region having the coding sequence shown in SEQ ID NO. 50.

In one or more embodiments, the intracellular domain of the costimulatory signaling molecule in the fusion protein is the IL-7Ralpha intracellular region, the coding sequence of which is shown in SEQ ID NO. 9.

In one or more embodiments, the intracellular domain of the costimulatory signaling molecule in the fusion protein is an IL-7Ralpha intracellular region mutant having the coding sequence shown in any one or more of SEQ ID NOs 13, 15, 17, 19.

In one or more embodiments, the fusion protein comprises a membrane surface tag comprising a BCMA extracellular domain having a coding sequence set forth in SEQ ID No. 21.

In one or more embodiments, the polynucleotide molecule is selected from any one of SEQ ID NOs 27-41, 52, 53, or is the complement of any one of the polynucleotide molecules shown.

The invention also provides a nucleic acid construct comprising a polynucleotide molecule according to any one of the embodiments of the invention.

In one or more embodiments, the nucleic acid construct is a vector.

In one or more embodiments, the vector is an expression vector or an integration vector; preferably a viral vector.

The invention also provides a genetically engineered cell expressing the fusion protein of any of the embodiments of the invention, and/or carrying the coding sequence of the fusion protein.

In one or more embodiments, the cell is an immune cell.

In one or more embodiments, the immune cells include T cells, NK cells, CAR-T, CAR-NK, TCR-T, CIK, NKT, and TIL.

In one or more embodiments, the cell also expresses a CAR, or carries a coding sequence for a CAR.

In one or more embodiments, the cell also expresses an exogenous TCR, or a coding sequence carrying an exogenous TCR.

The invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable adjuvant or the fusion protein, polynucleotide molecule, nucleic acid construct and genetically engineered cell according to any embodiment of the invention. The pharmaceutical composition is used for treating or preventing cancer.

In one or more embodiments, the cancer is melanoma.

The invention also provides the use of the fusion protein, the polynucleotide molecule, the nucleic acid construct and the genetically engineered cell according to any embodiment of the invention in preparing medicaments for treating or preventing cancers.

Drawings

Fig. 1: RTCA killing effect of TIL-CTRL, TIL-TBA-13 and TIL-TBA-16 on homologously paired melanoma primary tumor cells.

Fig. 2: in vivo killing effect of TIL-CTRL, TIL-TBA-13 and TIL-TBA-16 on homologously paired melanoma PDX tumor tissue.

Detailed Description

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute a preferred technical solution.

The invention uses the anti-TGF-beta antibody as the extracellular region of the signal conversion receptor, has higher affinity with TGF-beta factor compared with the signal conversion receptor based on the extracellular region of the TGF-beta receptor disclosed in WO2021244486A1, can activate the intracellular domain more effectively compared with the signal conversion receptor based on the extracellular region of the TGF-beta receptor, and further improves the activation and proliferation level of immune effector cells expressing the signal conversion receptor.

In the present invention, immune cells have the meaning well known in the art and refer to cells involved in or associated with an immune response, including lymphocytes, dendritic cells, monocytes/macrophages, granulocytes, mast cells, and the like. Lymphocytes include T lymphocytes, tumor-infiltrating lymphocytes, B lymphocytes, NK lymphocytes, and NKT cells. Immune cells suitable for use in the present invention include, inter alia, those typically used in adoptive cell therapy of tumors.

The immune cells of the invention express the signal transduction receptor of the invention and/or contain the coding sequence of the signal transduction receptor. The signal transduction receptors of the present invention are designed to bind inhibitory molecules but transmit a positive signal rather than an inhibitory signal. That is, these cells convert the "brake" signal to an "acceleration" signal to improve the anti-tumor effect of each immune cell.

Definition of the definition

The present invention uses the following terminology. For terms not specifically defined herein, they have meanings well known in the art.

The term "expression cassette" refers to the complete elements required for expression of a gene, including promoters, gene coding sequences, and PolyA tailing signal sequences.

The term "coding sequence" is defined herein as that portion of a nucleic acid sequence that directly determines the amino acid sequence of its protein product (e.g., signal transduction receptor, CAR). The boundaries of the coding sequence are typically determined by a ribosome binding site (for prokaryotic cells) immediately upstream of the open reading frame at the 5 'end of the mRNA and a transcription termination sequence immediately downstream of the open reading frame at the 3' end of the mRNA. Coding sequences may include, but are not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

The term "costimulatory signaling molecule" refers to a molecule that is present on the surface of an antigen-presenting cell and that is capable of binding to a costimulatory signaling molecule receptor on a Th cell to produce a costimulatory signal. It can activate the second signal of immune cell, strengthen the proliferation capacity of immune cell and the secretion function of cell factor, and prolong the survival time of activated immune cell. Proliferation of lymphocytes requires not only antigen binding but also signal of the co-stimulatory molecule. The co-stimulatory signal is transmitted to the T cell primarily through the co-stimulatory molecule CD80, CD86 expressed on the surface of the antigen presenting cell binding to the CD28 molecule on the surface of the T cell. B cells receive costimulatory signals through common pathogen components such as LPS, or through complement components, or through activated antigen-specific CD40L on Th cell surfaces.

The term "linker" or hinge is a polypeptide fragment that connects between different proteins or polypeptides in order to maintain the connected proteins or polypeptides in their respective spatial conformations in order to maintain the function or activity of the protein or polypeptide. Exemplary linkers include linkers comprising G and/or S, and for example Furin 2A peptides.

The term "pharmaceutically acceptable excipients" refers to carriers and/or excipients that are pharmacologically and/or physiologically compatible with the subject and active ingredient, which are well known in the art (see, e.g., remington's pharmaceutical sciences, improved by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995), and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers. For example, pH modifiers 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.

The term "effective amount" refers to the amount that achieves treatment, prevention, alleviation and/or relief of a disease or condition of the present invention in a subject.

The term "disease and/or disorder" refers to a physical state of the subject that is associated with the disease and/or disorder of the present invention.

The term "subject" may refer to a patient or other animal, particularly a mammal, such as a human, dog, monkey, cow, horse, etc., receiving a pharmaceutical composition of the invention to treat, prevent, ameliorate and/or alleviate a disease or disorder described herein.

The term "extracellular region" refers to the region of a membrane protein that is located outside of a cell.

The term "domain" refers to a region of a protein having a specific structure and independent function, the number of amino acid residues of a common domain being between 100 and 400, the smallest domain being only 40 to 50 amino acid residues, and the large domain being more than 400 amino acid residues.

Signal transduction receptor

The signal transduction receptor of the present invention is a fusion protein comprising a TGF-beta antibody or antigen binding fragment thereof fused to an intracellular domain (also referred to as an "intracellular region") of an immunostimulatory molecule (also referred to as a "costimulatory signaling molecule"). More specifically, the signal transduction receptor of the present invention includes a TGF-beta antibody or antigen binding fragment thereof as an extracellular region, a transmembrane region, and an intracellular domain of a costimulatory signal molecule.

In the present invention, "antibody" includes but is not limited to: monoclonal antibodies (including full length antibodies, which have an immunoglobulin Fc region), antibody compositions with multi-epitope specificity, multi-specific antibodies (e.g., bispecific antibodies), diabodies, single domain antibodies (sdabs), heavy chain antibodies (hcabs), nanobodies (nanobodies), minibodies (minibodies), and antibody fragments, particularly antigen-binding fragments, e.g., single chain antibodies (scFv), fab ', and F (ab') ₂ 。

"variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domains of the heavy and light chains may be referred to as "VH" and "VL", respectively. These domains are typically the most variable parts of an antibody (relative to other antibodies of the same type) and contain antigen binding sites.

The term "variable" refers to the case where certain segments in the variable domain differ widely in antibody sequence. The variable domains mediate antigen binding and define the specificity of a particular antibody for its particular antigen. However, variability is not evenly distributed across all amino acids spanned by the variable domains. Instead, it focuses on three segments called hypervariable regions (HVRs), both in the light and heavy chain variable domains, i.e., HCDR1, HCDR2, HCDR3 for the heavy chain variable region (which may be abbreviated as CDR1, CDR2, CDR3 in heavy chain antibodies) and LCDR1, LCDR2, and LCDR3 for the light chain variable region, respectively. The more highly conserved portions of the variable domains are called Framework Regions (FR). The variable domains of the natural heavy and light chains each comprise four FR regions (FR 1, FR2, FR3 and FR 4) that mostly take on a beta-folded conformation, linked by three HVRs that form a loop linkage and in some cases form part of a beta-folded structure. The HVRs in each chain are held together in close proximity by the FR regions and, together with the HVRs of the other chain, contribute to the formation of the antigen binding site of the antibody. Typically, the light chain variable region is of the structure FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4 and the heavy chain variable region is of the structure FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4. The constant domains are not directly involved in binding of antibodies to antigens, but exhibit a variety of effector functions, such as participation of antibodies in antibody-dependent cell-mediated cytotoxicity.

"Fc region" (crystallizable fragment region) or "Fc domain" or "Fc" refers to the C-terminal region of the antibody heavy chain that mediates binding of immunoglobulins to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or binding to the first component (C1 q) of the classical complement system. In IgG, igA and IgD antibody isotypes, the Fc region consists of two identical protein fragments from the CH2 domain and the CH3 domain of the two heavy chains of the antibody; the Fc region of IgM and IgE contains three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, a human IgG heavy chain Fc region is generally defined as the stretch of sequence from the amino acid residue at heavy chain position C226 or P230 to the carboxy-terminus, wherein the numbering is according to the EU index as in Kabat.

An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen-binding and/or variable regions of an intact antibody. The antibody fragment is preferably an antigen binding fragment of an antibody. Examples of antibody fragments include Fab, fab ', F (ab') ₂ And Fv fragments; a diabody; a linear antibody; a single chain antibody molecule; an scFv-Fc fragment; multispecific antibodies formed from antibody fragments; and any fragment that should be capable of increasing half-life by chemical modification or by incorporation into liposomes. Digestion of an antibody with papain produces two identical antigen-binding fragments, called "Fab" fragments, and one residual "Fc" fragment, the name of which reflects its ability to crystallize readily. The Fab fragment consists of the complete light chain and heavy chain variable domain (VH) and one heavy chain first constant domain (CH 1). Each Fab fragment is monovalent in terms of antigen binding, i.e. it has a single antigen binding site. Pepsin treatment of antibodies produced a larger F (ab') 2 fragment, roughly equivalent to two Fab fragments linked by disulfide bonds, with different antigen binding activities and still capable of cross-linking the antigen. Fab' fragments differ from Fab fragments by the addition of some additional residues at the carboxy terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. F (ab') ₂ Antibody fragments were initially generated as pairs of Fab 'fragments with hinge cysteines between the Fab' fragments. Other chemical couplings of antibody fragments are also known. The Fc fragment comprises the carboxy-terminal portions of two heavy chains held together by disulfide bonds. The effector function of antibodies is determined by sequences in the Fc region, which is also the region recognized by Fc receptors (fcrs) found on certain cell types.

"Fv" is the smallest antibody fragment that contains the complete antigen recognition and binding site. The fragment consists of a dimer of one heavy chain variable domain and one light chain variable domain in tight, non-covalent association. Six hypervariable loops (3 loops each for heavy and light chains) are highlighted from the fold of these two domains, contributing to the antigen-binding amino acid residues and conferring antigen-binding specificity to the antibody. However, even a single variable domain (or half Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although with less avidity than the complete binding site. "Single chain Fv" may also be abbreviated "sFv" or "scFv" and is an antibody fragment comprising the VH and VL domains of an antibody linked into one polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains such that the sFv forms the desired antigen-binding structure.

Antibodies herein also include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

"humanized" form of a non-human (e.g., murine) antibody refers to a chimeric antibody that minimally comprises sequences derived from a non-human immunoglobulin. Thus, a "humanized antibody" generally refers to a non-human antibody in which the variable domain framework regions are exchanged for sequences found in a human antibody. Typically in humanized antibodies, the entire antibody (except for the CDRs) is encoded by a polynucleotide of human origin or is identical to such an antibody (except for the CDRs). CDRs (some or all of which are encoded by nucleic acids derived from non-human organisms) are grafted into the beta-folding framework of the human antibody variable region to produce an antibody, the specificity of which is determined by the grafted CDRs. Methods for producing such antibodies are well known in the art, for example, using mice with genetically engineered immune systems.

"human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and/or produced using any of the techniques disclosed herein for producing a human antibody. This definition of human antibodies specifically excludes humanized antibodies that comprise non-human antigen binding residues. Human antibodies can be generated using a variety of techniques known in the art, including phage display libraries. The antibody of any one of the embodiments of the invention is a chimeric antibody or a fully human antibody; preferably fully human antibodies.

In certain embodiments, the HCDR1 of the anti-TGF-beta antibodies described herein is set forth in SEQ ID NO. 44. In certain embodiments, the HCDR2 of the anti-TGF-beta antibodies described herein is set forth in SEQ ID NO. 45. In certain embodiments, the HCDR3 of the anti-TGF-beta antibodies described herein is set forth in SEQ ID NO. 46. In certain embodiments, the LCDR1 of the anti-TGF-beta antibodies described herein is set forth in SEQ ID NO. 47. In certain embodiments, the LCDR2 of the anti-TGF-beta antibodies described herein is as set forth in SEQ ID NO. 48. In certain embodiments, the LCDR3 of the anti-TGF-beta antibodies described herein is set forth in SEQ ID NO. 49. In certain embodiments, the VH of the anti-TGF-beta antibodies described herein is set forth in SEQ ID NO. 42. In certain embodiments, the VL of an anti-TGF-beta antibody described herein is set forth in SEQ ID NO. 43.

Co-stimulatory signaling molecules in the present invention include CD28, CD134 (OX 40), CD137 (4-1 BB), LCK, ICOS, DAP, siglec-9, siglec-10, siglec-15, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2R, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21R, CD27, and CD40. One or more of these intracellular domains (intracellular regions) or functional fragments thereof of costimulatory signaling molecules or mutants that retain the costimulatory signaling molecules to deliver costimulatory signaling, activate the biological function of immune cells can be used to construct the signaling receptors of the invention. An exemplary IL-7R may be IL-7Ralpha, the exemplary amino acid sequence of which and the corresponding coding sequence of which are shown in SEQ ID NOS 10 and 9, respectively. The amino acid sequence of the intracellular region of exemplary CD28 and the corresponding coding sequence can be shown in SEQ ID NOS.8 and 7, respectively. The amino acid sequence of the intracellular region of exemplary CD134 and the corresponding coding sequence may be as shown in SEQ ID NOS.51 and 50, respectively. The intracellular domain of the costimulatory signaling molecule may also be that described in WO2021244486, which is incorporated herein by reference in its entirety.

In the present invention, the extracellular domain of an anti-TGF-beta antibody is linked to the intracellular domain of a costimulatory signal molecule through a transmembrane region (transmembrane domain). The transmembrane region may be of any origin. Suitable transmembrane regions for use in the present invention include, but are not limited to, any one or more of the transmembrane regions of CD28, CD134 (OX 40), CD137 (4-1 BB), LCK, ICOS, DAP, siglec-9, siglec-10, siglec-15, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2R, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21R, CD27, and CD40, or mutants thereof that retain transmembrane function. The transmembrane region may be from the same molecule as the intracellular domain or may be from a different molecule. In the present invention, the preferred transmembrane region is derived from the CD28 or IL-7Ralpha transmembrane region and mutants thereof. Exemplary amino acid sequences and coding sequences for the CD28 transmembrane region are shown in SEQ ID NOS.6 and 5, respectively. Exemplary amino acid sequences and coding sequences for the IL-7Ralpha transmembrane region are shown in SEQ ID NOS 12 and 11, respectively. Exemplary amino acid sequences of mutants of the IL-7Ralpha transmembrane region may be selected from any of SEQ ID NOs 14, 16, 18, 20; preferably, the coding nucleic acid sequence is selected from any one of SEQ ID NO. 13, 15, 17, 19. The transmembrane region may also be a mutant of the IL-7Ralpha transmembrane region described in WO2021244486, which is incorporated herein by reference in its entirety.

It is understood that "functional fragment" as used herein refers to a fragment that retains the desired biological function. For example, a functional fragment of an intracellular domain as described herein refers to a fragment that retains the biological function of the costimulatory signaling molecule to deliver a costimulatory signal, activating an immune cell. Functional fragments of each extracellular domain suitable for use in the present invention can be readily determined by one of skill in the art in combination with prior art means in the art.

The signal transduction receptor of the present invention may also have a membrane surface tag extracellular. Thus, in some embodiments, the extracellular domains described herein comprise the anti-TGF-beta antibody and a membrane surface tag. Herein, "membrane surface tag" includes BCMA extracellular domain or fragment thereof. Preferably, the BCMA ectodomain comprises the sequence shown in SEQ ID No. 22; the nucleic acid sequence comprises the sequence shown in SEQ ID NO. 21. The membrane surface tag may function as an immune braking element, recognition element, linker, element that induces ADCC, ADCP and/or CDC effects.

The membrane surface tag may also have a linker fragment at the N-terminus or C-terminus of the BCMA extracellular domain for linking to other polypeptides or polypeptide portions. The connecting segments are typically hinge regions or linkers. Exemplary linkers include the sequences shown as SEQ ID NOS.24 or 26, and the nucleic acid sequences shown as SEQ ID NOS.23 or 25, respectively. The hinge region includes one or more selected from the group consisting of: an extracellular hinge region of CD8, an IgG1 Fc CH2CH3 hinge region, an IgD hinge region, a CD28 extracellular hinge region, an IgG4 Fc CH2CH3 hinge region, and an extracellular hinge region of CD 4.

In some embodiments, the BCMA extracellular domain is linked to the C-terminus of the anti-TGF-beta antibody shown in SEQ ID NO. 4 by a linker shown in SEQ ID NO. 24; or the BCMA extracellular domain is linked to the N-terminus of the anti-TGF-beta antibody shown in SEQ ID NO. 4 through a linker shown in SEQ ID NO. 26.

The "mutant" as referred to herein includes mutants of each antibody, transmembrane region and intracellular domain, as long as the mutants retain the respective biological functions of the antibody, transmembrane region and intracellular domain. For example, mutants of antibodies suitable for use in the present invention include mutants having at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity to the antibody used as a control; mutants suitable for use in the transmembrane region of the invention include mutants having at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity to the transmembrane region as a comparison; mutants of the intracellular domains suitable for use in the present invention include mutants having at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity to the intracellular domain to be compared. Alternatively, the mutants of the present invention have one or more (e.g., 20 or less, 15 or less, 10 or less, 8 or less, 5 or less, or 3 or less, e.g., 1-20, 1-10, etc.) amino acid residues inserted, substituted or deleted as compared to the sequences used as a comparison. For example, conservative substitutions with amino acids that are similar or analogous in nature typically do not alter the function of the protein or polypeptide. "similar or analogous amino acids" include, for example, families of amino acid residues with similar side chains, including amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

The invention also includes mutants of the signal transduction receptor described above, such as mutants having at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity to the signal transduction receptor. More specifically, the present invention includes mutants having one or more (e.g., 20 or less, 15 or less, 10 or less, 8 or less, 5 or less, or 3 or less, e.g., 1-20, 1-10, etc.) amino acid residues inserted, substituted or deleted as compared to the signal transduction receptor described above. Such mutants retain the biological function of the signal transduction receptor of the present invention, including but not limited to converting a signal recognizing TGF-beta to a stimulatory signal enhancing immune cell proliferation. Mutations can occur in any, any two, or all three of the extracellular domains, transmembrane regions, and intracellular domains described herein.

The polypeptides described herein may be modified polypeptides. Modified (typically without altering the primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications during synthesis and processing of the polypeptide or during further processing steps. Such modification may be accomplished by exposing the polypeptide to an enzyme that performs glycosylation (e.g., mammalian glycosylase or deglycosylase). Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides modified to improve their proteolytic resistance or to optimize solubility.

In one or more embodiments, the fusion protein comprises, from N-terminus to C-terminus: an anti-TGF-beta single chain antibody with VH shown as SEQ ID NO. 42 and/or VL shown as SEQ ID NO. 42, a CD28 transmembrane region shown as SEQ ID NO. 6, an IL7Ralpha transmembrane region shown as SEQ ID NO. 12 or an IL7Ralpha transmembrane region mutant shown as any one of SEQ ID NO. 14, 16, 18, 20, and a CD28 intracellular domain shown as SEQ ID NO. 8, an IL7Ralpha intracellular domain shown as SEQ ID NO. 10 or a CD134 intracellular domain shown as SEQ ID NO. 51.

In one or more embodiments, the fusion protein comprises, from N-terminus to C-terminus: an anti-TGF-beta single chain antibody having a VH as shown in SEQ ID NO. 42 and/or a VL as shown in SEQ ID NO. 42, a linker as shown in SEQ ID NO. 24 or a linker as shown in SEQ ID NO. 26, a BCMA extracellular domain as shown in SEQ ID NO. 22, a CD28 transmembrane region as shown in SEQ ID NO. 6, an IL7Ralpha transmembrane region as shown in SEQ ID NO. 12 or an IL7Ralpha transmembrane region mutant as shown in any one of SEQ ID NO. 14, 16, 18, 20, and a CD28 intracellular domain as shown in SEQ ID NO. 8 and/or a CD134 intracellular domain as shown in SEQ ID NO. 51.

In one or more embodiments, the fusion protein comprises, from N-terminus to C-terminus: an anti-TGF-beta single chain antibody as shown in SEQ ID No. 22, a linker as shown in SEQ ID No. 24 or a linker as shown in SEQ ID No. 26, a VH as shown in SEQ ID No. 42 and/or a VL as shown in SEQ ID No. 42, a CD28 transmembrane region as shown in SEQ ID No. 6, an IL7Ralpha transmembrane region as shown in SEQ ID No. 12 or an IL7Ralpha transmembrane region mutant as shown in any one of SEQ ID No. 14, 16, 18, 20, and a CD28 intracellular domain as shown in SEQ ID No. 8, an IL7Ralpha intracellular domain as shown in SEQ ID No. 10 or a CD134 intracellular domain as shown in SEQ ID No. 51.

Exemplary signaling receptors of the invention include, but are not limited to, signaling receptors comprising or consisting of the extracellular domains, transmembrane regions, and intracellular domains shown in each row of table 1 below:

TABLE 1

In some embodiments, the signal transduction receptors described herein further comprise a signal peptide. Preferably, the signal peptide is located at the N-terminus of the signal transduction receptor. The signal peptide may be any signal peptide capable of directing the polypeptide out of the core as is conventional in the art, including but not limited to CD8, CD4, CD28, CD137, EGFR, TGFBRI, TGFBRII, TGFBRIII and light chain signal peptides. In some embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO. 2, the coding sequence of which is set forth in SEQ ID NO. 1.

It will be appreciated that the extracellular domains and transmembrane regions, and/or the transmembrane regions and intracellular domains described herein may be linked by a linker sequence, as desired. Linker sequences known in the art, such as those containing G and S, such as (GSSS) n or (GSSSs) n, where n is an integer from 1 to 8, may be used.

The immune cells of the invention may further express a CAR, or contain a coding sequence for a CAR. The CARs of the present invention may be a variety of CARs well known in the art.

The CAR may in turn comprise a polypeptide that binds to a tumor cell membrane antigen (e.g., scFv), a hinge region, a transmembrane region, and an intracellular signaling region. The CARs of the invention can be constructed using hinge, transmembrane and intracellular signal regions well known in the art for constructing CARs. In general, polypeptides that bind tumor cell membrane antigens are capable of binding with moderate affinity to membrane antigens that are widely expressed by tumor cells, and are typically inserted with an epitope at a position selected from any 1, 2 or 3 of the following 3 positions: the N-terminus of the polypeptide, between the polypeptide and the hinge region, and within the polypeptide. The polypeptide combined with the tumor cell membrane antigen is a natural polypeptide or an artificial synthetic polypeptide; preferably, the synthetic polypeptide is a single chain antibody or Fab fragment.

The chimeric antigen receptor of the invention may be directed against one or more of the following antigens: CD19, CD20, CEA, GD2 (also known as B4GALNT 1), FR (also known as Flavin reductase), PSMA (also known as prostate specific membrane antigen), PMEL pre-melanosome protein), CA9 (carbonic anhydrase IX), CD171/L1-CAM, IL-13RL1, MART-1 (also known as mucin-A), ERBB2, NY-ESO-1 (also known as CTAG1B, cancer/testis antigen 1B), MAGE (melanoma associated antigen E1) family proteins, BAGE (B melanoma antigen family) family proteins, GAGE (growth hormone releasing factor) family proteins, AFP, MUC1 (also known as mycin 1), CD22, CD23, CD30, CD33, CD44v7/8, CD70, VEGFR1, VEGFR2, IL-11R/, EGP-2, EGP-40, FBP, GD3 (also known as SIA 1), PSCA (prostate stem cell antigen), KLA (PSA 9), GAGE (also known as GRGE protein) family proteins, GAGE (growth hormone releasing factor) family proteins, AFP, MUC1 (also known as MUC 1), CD22, CD23, CD30, CD33, CD44v7/8, CD70, VEGFR1, VEGFR2, IL-11R/, EGP-2, EGP-40, GD3 (also known as FBST 8SIA 1), PSCA (prostate stem cell antigen), PSCA (PSA 9), PSA 3, GAGE 3, GRBB 3 (Grave 3, GRCA 3, and GLP 3 (Grave 3) type 3, GRCA 1).

A single cell may express multiple CARs, including CARs targeting different tumor antigens.

T Cell Receptor (TCR) -T

The immune cells of the invention may further express an exogenous TCR or contain a coding sequence that expresses an exogenous TCR gene. The TCRs of the present invention may be any known in the art, for example, HLA-matched TCRs, known in sequence and structure, and known in combination with antigen peptide sequences.

The exogenous TCRs described herein include αβ double chains that can form complete TCR complexes with double-stranded structures of γε, δε, and ζζ expressed endogenously by immune effector cells such as T cells. The exogenous gene encoding the exogenous TCR of the invention includes an alpha beta double-stranded gene, and the alpha chain and beta chain coding sequences are covalently linked by a linker sequence that can be cleaved in vivo, such as the coding DNA sequence of P2A, T A or F2A sequences, or by a DNA fragment encoding an IRES sequence. In addition to the αβ duplex encoding the exogenous TCR, the gene encoding the exogenous TCR of the present invention may comprise a tag protein gene, such as EGFP, RFP, YFP gene, or the like, expressed in fusion with the αβ gene. The tag protein gene may be covalently linked to the αβ double stranded gene by a linker sequence that can be cleaved in vivo, such as a 2A sequence, e.g., a DNA sequence encoding P2A, T2A or F2A, or by a DNA sequence encoding an IRES sequence. The tag protein, such as EGFP, RFP, YFP gene, which is expressed together with TCR alpha beta double chain, can be used as identification index for detecting exogenous TCR expression.

The TCR-T of the invention may be directed against one or more of the following antigens: CD19, CD20, CEA, GD2 (also known as B4GALNT 1), FR (also known as Flavin reductase), PSMA (also known as prostate specific membrane antigen), PMEL pre-melanosome protein), CA9 (carbonic anhydrase IX), CD171/L1-CAM, IL-13RL1, MART-1 (also known as mucin-A), ERBB2, NY-ESO-1 (also known as CTAG1B, cancer/testis antigen 1B), MAGE (melanoma associated antigen E1) family proteins, BAGE (B melanoma antigen family) family proteins, GAGE (growth hormone releasing factor) family proteins, AFP, MUC1 (also known as mycin 1), CD22, CD23, CD30, CD33, CD44v7/8, CD70, VEGFR1, VEGFR2, IL-11R/, EGP-2, EGP-40, FBP, GD3 (also known as GD 8SIA 1), PSCA (prostate stem cell antigen), KLA (FSK 9), GAGE (also known as GAGE) family proteins, GAGE (growth hormone releasing factor) family proteins, AFP, MUC1 (also known as MUC 1), CD22, CD23, CD30, CD44v7/8, CD70, VEGFR1, VEGFR2, IL-11R/, EGFR2, EGP-40, FBP, GD3 (also known as GL 8SIA 1), PSCA (precursor 1), KLA (F3), GRCA 3 (Grave 3), GRCA 3, GRCA 1) and GRCA 1 (Grave 3, GRCA 1).

A single cell may express multiple exogenous TCRs, including exogenous TCRs targeting different tumor antigens.

Polynucleotide molecules

The present invention provides polynucleotide molecules encoding the signal transduction receptors described herein. The invention also provides the complementary sequence of the coding sequence of the signal transduction receptor. The polynucleotide molecule may be a recombinant nucleic acid molecule or may be synthetic; it may comprise DNA, RNA and PNA (peptide nucleic acid) and may be a hybrid thereof.

Also provided is an expression cassette for a signal transduction receptor of the present invention, which is a nucleic acid construct comprising a promoter, a signal transduction receptor coding sequence, and a PolyA tailing signal sequence. Other elements required for expression may also be included in the nucleic acid construct, including but not limited to enhancers and the like.

Also provided is a vector comprising a polynucleotide molecule, expression cassette or nucleic acid construct described herein. Vectors may be plasmids, cosmids, viruses, and phages. The vector may be a cloning vector or an expression vector. The expression vector may be a transposon vector. In certain embodiments, the expression vector is one or more selected from the group consisting of: piggybac, sleep reliability, frog priority, tn5 and Ty. In addition to the polynucleotide molecules of the invention, the expression vectors will typically contain other elements typically contained in vectors, such as multiple cloning sites, resistance genes, replication initiation sites, and the like. In certain embodiments, the recombinant expression vector employs pUC18, pUC19, pMD18-T, pMD19-T, pGM-T, pUC57, pMAX or pDC315 series vectors as the backbone. In other embodiments, the recombinant expression vector employs a pCDNA3 series vector, a pCDNA4 series vector, a pCDNA5 series vector, a pCDNA6 series vector, a pRL series vector, a pUC57 vector, a pMAX vector, or a pDC315 series vector as a backbone. In certain embodiments, the invention uses the pNB vector constructed by CN105154473 a. In certain embodiments, the invention uses the pKB20 vector described in WO2022078310 A1.

The CARs of the invention may also be expressed in the immune cells of the invention by conventional vectors. The vector may be a conventional CAR-expressing vector, including but not limited to the various transposon vectors and recombinant expression vectors described previously.

In some embodiments, the same vector encodes both the signaling receptor and CAR of the invention. The vector may be a bicistronic. The coding sequence of the CAR may be disposed 5 'or 3' to the signal transduction receptor coding sequence. Expression of the CAR and signaling receptor may be under the direction of the same or different regulatory sequences.

Where the polynucleotide sequence is known, each polynucleotide molecule may be prepared by methods conventional in the art and the corresponding vector constructed. Recombinant vectors can be constructed using methods well known to those skilled in the art, see, for example, sambrook et al, ausubel (1989), or other standard textbook techniques. Alternatively, the nucleic acid molecules and vectors can be reconstituted into liposomes for delivery to target cells. Vectors containing the nucleic acid molecules of the invention may be transferred into host cells by well known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly used for prokaryotic cells, whereas calcium phosphate treatment or electrotransfection may be used for other cellular hosts, see Sambrook.

Host cells

Herein, when expressing a heterologous nucleic acid sequence, "host cell" refers to a eukaryotic cell that is capable of replicating the vector and/or expressing the heterologous gene encoded by the vector. Host cells can be used as acceptors for vectors. The host cell may be "transfected" or "transformed," which refers to the process by which exogenous nucleic acid is transfected or transduced into the host cell. Transformed cells include primary subject cells and their progeny. The terms "engineered" and "recombinant" cells or host cells as used herein often refer to cells into which exogenous nucleic acid sequences, such as vectors, have been introduced. Thus, recombinant cells can be distinguished from naturally occurring cells that do not contain the introduced recombinant nucleic acid.

Herein, host cells include cells carrying the polynucleotide molecules and/or polypeptides described herein. In particular, the invention provides cells carrying the signal transduction receptor and/or the coding sequence thereof of the invention. The cells of the invention are preferably immune cells and can be used for adoptive cell therapy of tumors. Such cells of the invention are also referred to as cells modified by the signal transduction receptor of the invention.

More specifically, the cells of the invention are preferably immune effector cells, including T cells, such as cytotoxic T cells (also known as TC, cytotoxic T lymphocytes, CTLs, T killer cells, cytolytic T cells, cd8+ T cells, or killer T cells), NK cells, NKT cells, CAR-T, CAR-NK, TCR-T, CIK, TIL; and other immune cells that can elicit effector functions.

Herein, cells may be autologous cells, syngeneic cells, allogeneic cells, and even in some cases xenograft cells, relative to the individual receiving them.

The nucleic acid construct/recombinant expression vector of the invention may be transferred into a cell of interest. Methods of transfer are conventional in the art and include, but are not limited to: viral transduction, microinjection, particle bombardment, gene gun transformation, electrotransformation, and the like. In certain embodiments, the nucleic acid construct or recombinant expression vector is electrotransferred.

In addition to carrying the signal transduction receptor and/or coding sequences thereof of the present invention, the cells of the present invention may have one or more additional properties useful in cellular immunotherapy (e.g., adoptive cell therapy for tumors). Such other properties may be inherent to the cell or may be part of the cell after genetic manipulation by a human. For example, the cells of the invention may carry chimeric antigen receptors, alphabetaT cell receptors, and/or antigen-specific receptors, such as tumor-specific receptors, or coding sequences thereof.

Pharmaceutical composition

Herein, "pharmaceutical composition" refers to a composition for administration to an individual and encompasses a composition of cells for immunotherapy. The pharmaceutical compositions of the invention may also comprise a pharmaceutically acceptable carrier. Examples of suitable pharmaceutical carriers are known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions, and the like. Compositions comprising such carriers can be formulated by well-known conventional methods. These pharmaceutical compositions may be administered to a subject in a suitable dosage.

The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, the dosage for any one patient depends on a variety of factors including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs administered simultaneously.

The compositions of the present invention may be administered locally or systemically. In certain embodiments, the compositions provided herein (e.g., cells expressing the signaling receptors described herein) may be administered parenterally, e.g., intravenously, intraarterially, intrathecally, subcutaneously, or intramuscularly. In certain other embodiments, DNA encoding the constructs provided herein may be administered directly to a target site, for example, delivered to an internal or external target site by a gene gun or to an intra-arterial site by a catheter. In a preferred embodiment, the pharmaceutical composition is administered subcutaneously, and in a more preferred embodiment, intravenously. Parenteral formulations include sterile aqueous or nonaqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous vehicles include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral carriers include sodium chloride solution, lin Geyou dextrose, dextrose and sodium chloride, ringer's lactate solution or fixed oils. Intravenous vehicles include liquid and nutritional supplements, electrolyte supplements (such as those based on Yu Linge dextrose), and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like. Furthermore, the pharmaceutical composition of the invention may comprise a proteinaceous carrier, such as serum albumin or an immunoglobulin, preferably of human origin. In addition to the proteinaceous chimeric cytokine receptor construct or nucleic acid molecule or vector encoding the same, it is contemplated that the pharmaceutical composition of the invention may also comprise a biologically active agent, depending on the intended use of the pharmaceutical composition.

Compositions for parenteral (e.g., intravenous) administration of the cells described herein may also be stored in lyophilized form or in solution (e.g., lyophilized formulations). The lyophilized formulation may be stored in a ready-to-use form or in a form that is further formulated prior to administration. The cryopreservation formulation can withstand long distance transport without damaging the cells. In addition to the cells themselves, cryopreservation formulations typically include components such as cell cryopreservation solution, human Serum Albumin (HSA), and the like. Prior to administration (e.g., intravenous infusion), the cryopreserved pharmaceutical composition is stored (e.g., in liquid nitrogen). The frozen preparation can be directly infused into a patient or formulated as an infusion composition after thawing. The composition and concentration of conventional frozen stock solutions are known to those skilled in the art. For example, the frozen stock solution or infusion composition may further comprise dimethylsulfoxide, sodium chloride, glucose, sodium acetate, potassium chloride, magnesium chloride, or the like, the concentration of which may be determined by one of skill in the art (e.g., an experienced physician) depending on the condition of the cell, disease, patient, or the like.

Method and application

The signal conversion receptor, the polynucleotide molecule, the vector, the host cell and the pharmaceutical composition containing the same can be used for preventing, treating or relieving cancers, especially cancers with corresponding tumor antigens expressed on the surfaces of cancer cells, or used for preparing medicines for preventing, treating or relieving cancers.

As used herein, "treatment" or "treatment" includes any beneficial or desired effect on the symptoms or lesions of a disease or pathological condition, and may include even a small reduction in one or more measurable markers of the disease or condition under treatment (e.g., cancer). Treatment may optionally include a reduction or alleviation of symptoms of the disease or disorder, or a delay in the progression of the disease or disorder. "treating" does not necessarily mean complete eradication or cure of a disease or disorder or associated symptoms thereof.

"preventing" as used herein refers to a method for preventing, inhibiting, or reducing the likelihood of occurrence or recurrence of a disease or disorder (e.g., cancer). It also refers to delaying the onset or recurrence of a disease or disorder or delaying the onset or recurrence of symptoms of a disease or disorder. As used herein, "preventing" also includes reducing the intensity, impact, symptoms and/or burden of a disease or disorder before it occurs or recurs.

The invention includes the administration of cells, polynucleotide molecules and vectors, alone or in any combination, using standard vectors and/or gene delivery systems, optionally together with pharmaceutically acceptable carriers or excipients. In certain embodiments, the polynucleotide molecule or vector may be stably integrated into the genome of the subject following administration.

In particular embodiments, viral vectors that are specific for certain cells or tissues and persist in the cells may be used. Suitable pharmaceutical carriers and excipients are well known in the art. The compositions prepared according to the invention may be used to prevent or treat or delay the diseases identified above.

Furthermore, the present invention provides a method for preventing, treating or alleviating cancer, comprising the steps of: administering to a subject in need thereof an effective amount of a cell carrying a signaling receptor, polynucleotide molecule, and/or vector described herein and/or produced by a method described herein.

The methods herein can be used to prevent, treat, or ameliorate a variety of cancers, including various solid and hematological tumors, including but not limited to lung cancer (e.g., non-small cell lung cancer), colon cancer, cervical cancer, liver cancer, fibrosarcoma, erythroleukemia, prostate cancer, breast cancer, pancreatic cancer, ovarian cancer, melanoma, and glioma, among others. More specifically, cancers herein include, but are not limited to, breast, prostate, lung, and colon cancer or epithelial cancers, such as breast cancer, colon cancer, prostate cancer, head and neck cancer, skin cancer, melanoma; genital-urinary tract cancers, such as ovarian cancer, endometrial cancer, cervical cancer; renal cancer, lung cancer, gastric cancer, small intestine cancer, liver cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, esophageal cancer, salivary gland cancer, thyroid cancer, etc. Administration of the compositions of the invention may be useful for all stages and types of cancer, including for example, minimal residual disease, early stage cancer, advanced cancer, and/or metastatic cancer, and/or cancer that is refractory to treatment.

By way of example, a cancer patient or a patient susceptible to cancer or a patient suspected of having cancer is treated as follows. The modified cells as described herein may be administered to an individual and left for an extended period of time. The individual may receive one or more administrations of cells, and the interval between administrations may be days, weeks, months or years. In particular embodiments, multiple administrations may occur over weeks or months, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more weeks or months. In some embodiments, the genetically modified cells are encapsulated to inhibit immune recognition and are located at a tumor site. In the case where cells are provided to an individual after tumor recurrence following initial treatment with cells of the invention, the cells may be altered to recognize different target tumor antigens. For example, where an initial round includes cells carrying a signaling receptor of the invention and another receptor specific for a particular antigen, the receptor for a different particular antigen may be used after a subsequent round (including after tumor recurrence).

In some embodiments, an effective amount of therapeutic cells carrying or expressing the signaling receptor of any of the embodiments of the invention and optionally a CAR or exogenous transgenic TCR is provided to an individual in need thereof. These cells may be delivered simultaneously or non-simultaneously with one or more other cancer treatments. These cells and other cancer therapeutic agents may be delivered in the same or separate formulations. Cells and other cancer therapeutic agents may be provided to an individual by separate delivery routes. Cells and/or other cancer therapeutic agents may be delivered by injection or intravenous or oral administration, for example, at a tumor site. Conventional delivery routes for such compositions are known in the art.

The number of cells employed will depend on a variety of circumstances, such as the purpose of the introduction, the lifetime of the cells, the regimen to be used, the number of administrations, the ability of the cells to reproduce, the stability of the recombinant construct, etc.

Cells may be administered as desired. In some embodiments, a variety of schemes may be used to adjust the scheme parameters. In particular embodiments, the route or number or timing of administration, the lifetime of the cells, and/or the number of cells present may vary. The number of administrations may depend, for example, at least in part, on the factors described above.

Kit for detecting a substance in a sample

Any of the compositions described herein may be included in a kit. In one non-limiting example, cells expressing a signaling receptor according to any of the embodiments of the invention and/or agents producing one or more cells for use in cell therapy comprising a recombinant expression vector may be included in a kit for use in cell therapy. The kit components are provided in a suitable container format.

Some of the components of these kits may be packaged in an aqueous matrix or in lyophilized form. The container means of these kits typically comprise at least one vial, test tube, flask, bottle, syringe or other container means in which the component may be placed and preferably appropriately dispensed. In the case where more than one component is present in the kit, the kit will typically also contain a second, third or other container in which the other components may be separately placed. However, various combinations of components may be included in the vial. The kits of the invention will typically also comprise means for containing the components in a commercially available closed constraint format. Such containers may include injection molded or blow molded plastic containers, wherein the desired vials are retained.

When the components of the kit are provided in one and/or more liquid solutions, the liquid solutions are aqueous solutions, particularly preferably sterile aqueous solutions. In some cases, the container means may itself be a syringe, pipette, and/or other such device.

The components of the kit may also be provided in dry powder form. When the reagents and/or components are provided as dry powders, the powders may be reconstituted by the addition of a suitable solvent. Thus, the kit may further comprise a second container means comprising a sterile, pharmaceutically acceptable buffer and/or other diluent.

In a specific embodiment of the invention, the cells to be used in the cell therapies described herein are provided in a kit. In some embodiments, the cell is essentially the only component of the kit. The kit may contain reagents and materials for preparing the desired cells. In particular embodiments, the reagents and materials comprise primers, nucleotides, suitable buffers or buffer reagents, salts, and the like for amplifying the desired sequence, and in some cases, the reagents comprise DNA and/or vectors encoding the signal transduction receptor and/or regulatory elements thereof described in any of the embodiments herein.

Embodiments of the present invention will be described in detail below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The specific techniques or conditions are not noted in the examples, and are carried out according to techniques or conditions described in the literature in the art (for example, refer to J. Sam Brookfield et al, guidelines for molecular cloning experiments, third edition, scientific Press, et al), corresponding references, or according to the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Examples

Example 1: construction of Signal conversion receptor expression vectors

The pKB20 vector was constructed according to the method described in example 1 on page 21 of the specification of PCT application WO2022078310A 1. According to the method for constructing pKB20-EGFP described in this example, a pKB20 vector containing the expression cassette of the foreign gene TBA was constructed. Specifically, the sequences shown in SEQ ID NOS 27-41 and 52-53 were obtained by the company, and the 2-terminal of the sequences shown in SEQ ID NOS 27-41 and 52-53, respectively, were ligated with a linker containing a corresponding cleavage site by using a ligase, and cloned into the prepared pKB20 vector according to the method described in example 1 on page 21 of WO2022078310A1 specification, designated pKB20-TBA-1, pKB20-TBA-2, pKB20-TBA-3 … … pKB20-TBA-17, respectively. The recombinant plasmids obtained above were transformed into E.coli (DH 5 c), and after sequencing was correct, plasmids were extracted and purified using the plasmid purification kit from Qiagen, to obtain high-quality plasmids for each recombinant expression vector. The structure and sequence of TBA-1-TBA-17 are shown in Table 2.

TABLE 2 TBA Signal transduction receptor Structure

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Example 2: isolated culture of melanoma tissue-derived TIL cells

Freshly excised melanoma specimens were collected and immediately treated under sterile conditions. The melanoma tissue of this example was treated and cultured to obtain TIL according to the medium described in example 1 of WO2022111571A1 and the tumor sample treatment method and TIL culture method described in example 2. WO2022111571A1 is incorporated by reference in its entirety.

The method comprises the following steps:

1) Preparing physiological saline containing 100U/mL penicillin, 100 mug/mL streptomycin and 50 mug/mL gentamicin for later use;

2) Placing the obtained tumor tissue sample of the freshly isolated tumor patient in a 10cm culture dish added with 30mL of the physiological saline prepared in the step 1) in a sterile environment in a secondary biosafety cabinet for washing, transferring the tumor tissue sample to a new 10cm dish added with 30mL of the physiological saline prepared in the step 1) for washing, and repeating the washing for 3 times;

3) Removing fat tissue and necrotic tissue with a sterile scalpel, cutting tumor tissue becomes a diameter of 3X 3mm ³ 2G-REX 100 culture tanks (available from Wilsonwolf), 42 randomly selected tumor tissue pieces were placed in each G-REX100 culture tank, seed cell culture medium was added to the culture tank, and the seed culture medium had the following composition: 3000IU/mL IL-2, 20ng/mL IL-7, 20ng/mL IL-15, 500U/mL GM-CSF, 1000IU/mL IFN-gamma, 3 μg/mL anti-CD 137mAb, 3 μg/mL anti-CD 28 mAb, 3 μg/mL anti-PD-1 mAb, 10ng/mL TNF-alpha, 5% v/v human AB serum, 1 XPS diabody, and a final volume of X-VIVO 15 basal medium; the redundant tumor tissue blocks are frozen and stored by a cryo-Stor 10 (purchased from BioLifeSolons) frozen solution through a program cooling instrument liquid nitrogen;

4) 3) adding 1L of the seed cell culture medium into a G-REX100 culture tank containing tumor tissue blocks, and adding 5% CO at 37 ℃ to the tumor tissue blocks ₂ Culturing, removing half of old seed cell culture medium every 4 days, supplementing half of fresh seed cell culture medium, centrifuging at 12 th day, and counting total number and activity rate of cells after harvesting TIL seed cells;

5) Taking the seed cells harvested in 4), re-suspending to 5.0X10 s with an expanding medium containing 500IU/mL IL-2, 7ng/mL IL-7, 30ng/mL IL-15, 5% v/v human AB serum, 1 XPS diabody and a final volume of X-VIVO 15 basal medium ⁵ Per mL, in a cell culture vessel pretreated with anti-CD 3 mAb, anti-CD 28 mAb and anti-CD 137 mAb coating, 37℃5% CO ₂ After 2 days of activation, the activated cells were collected by centrifugation and inoculated into a G-REX500M culture tank to which an expansion medium which had been preheated in advance was added, and the expansion medium in the G-REX500M culture tank was identical to the expansion medium described above. Each G-The volume of the expanded medium in REX500M was 5L. Activated seed cells were grown according to 2.5X10 ⁵ /cm ² Inoculation density inoculation of (C) 37℃ 5% CO ₂ Culturing, removing half volume of old expansion medium after cell count every 4 days, and supplementing half volume of fresh expansion medium until total cell count in each G-REX500M tank reaches 1.0X10 ¹⁰ Afterwards, the flasks were separated at a ratio of 1:2, and each flask was supplemented with fresh expansion medium to 5L and then continued to culture. Cells were harvested after a total of 12 days of culture in an enlarged medium of a G-REX500M culture tank before and after culturing to obtain TIL.

Example 3: genetic modification and proliferation of TIL

1) AIM-V medium was previously added to 12-well plates for a total of 18 wells, 2mL per well, and then transferred to a cell incubator at 37℃with 5% CO ₂ Preheating for 1 hour;

2) The ratio of the electrotransport liquid with single dosage per hole is carried out according to the following table:

	100μL Nucleocuvette ^TM Strip(μL)
		Nucleofector ^TM volume of solution	82
Electrolysis supplementary solution	18

Plasmids pKB20-TBA-1, pKB20-TBA-2, pKB20-TBA-3 … … pKB20-TBA-17 and control empty plasmid pKB20 tested as required, experimental group electrotransport system 17 and control group 1 were prepared;

3) Taking out the fruitThe TIL obtained in example 2 was transferred to 18 EP tubes, each of which was charged with 5X 10 ⁶ Centrifuging at 1200rpm for 5min, discarding supernatant, subsequently re-suspending cells with 500 μl physiological saline, and repeating the centrifugation step to wash cell pellet;

4) Adding plasmids pKB20-TBA-1, pKB20-TBA-2, pKB20-TBA-3 … … pKB20-TBA-17 and control empty plasmid pKB20 mug into the electrotransfer solution of each of the different experimental groups and the control groups prepared in the step 2), and standing at room temperature for less than 30 min;

5) Resuspension of all EP tubes with plasmid-containing electrotransfer solution prepared in 4), 100. Mu.L of each tube, carefully pipetting the cell resuspension into a LONZA 100. Mu.L electrotransfer cup, placing the electrotransfer cup into LONZA Nucleofector ^TM 2b, starting an electric transfer program in the electric transfer groove, wherein the electric transfer program selects X001;

6) After completion of electrotransfer, the electrotransfer cup was carefully removed, the cell suspension was aspirated and transferred to EP tubes, 200. Mu.L of pre-warmed AIM-V medium was added to each tube, followed by transfer to 1) wells containing pre-warmed AIM-V medium in 12-well plates, 37℃and 5% CO ₂ Culturing; after 5 days of culture, the expanded culture in separate bottles was continued to obtain TIL cells overexpressing TBA-1-TBA-17 and TIL cells of the control group, designated TIL-TBA-1, TIL-TBA-2 … … TIL-TBA-17 and TIL-CTRL, respectively.

Example 4: electrotransport TBA signaling receptor TIL phenotype assay

1. Cell viability and positivity of electrotransport TBA signaling receptor TIL

Cell viability was measured for each group by trypan blue staining and cell counter counting. The results show that the cell viability of TIL-TBA-1-TIL-TBA-17 and TIL-CTRL prepared in example 3 is above 93%.

The fluorescence labeling method is used for labeling the electrotransport TBA signal conversion receptor TIL, and the flow cytometry is used for detecting the cell ratio positive to the TBA signal conversion receptor gene expression, and the method is as follows:

1) Collection of TIL-TBA-1-TIL-TBA-17 and TIL-CTRL groups of cells, 1X 10 of cells were collected per group ⁶ 800g, centrifuging for 3min;

2) Discarding the supernatant, adding physiological saline into each cell sample to re-suspend the cells, and centrifuging for 3min, wherein 800 g;

3) The supernatant was discarded, and 100. Mu.L of physiological saline was added to each cell sample to resuspend the cells, and 2. Mu.L of biotin-conjugated TGF-. Beta.1 factor (available from acrobiosystems) was added to each tube; cargo number: TG 1-H8217), 30 min incubation at room temperature; 800g, centrifuging for 3min, and washing twice;

4) The supernatant was discarded, 100. Mu.L of physiological saline was added to each tube of pellet, and the cells were resuspended, and 2. Mu.L of PE-Streptavidin (PE strepitavidins, available from BDbiosciences; cargo number: 554061 30 min, 800g, centrifuging for 3min, washing twice, and discarding the supernatant;

5) Resuspension with 400 μl of physiological saline, and detection by up-flow cytometry.

Meanwhile, regarding to TIL (TIL-TBA-12-TIL-TBA-17) expressing a TBA signaling receptor containing an extracellular domain of BCMA, the extracellular domain of BCMA is used as a label, and the ratio of BCMA positive cells is detected by a flow cytometer by a direct labeling method, as follows:

1) Collection of TIL-TBA-12-TIL-TBA-17 Each group of cells, 1X 10 of the number of cells per group was collected ⁶ 800g, centrifuging for 3min;

2) Discarding the supernatant, adding physiological saline to resuspend cells, and centrifuging for 3min at 800 g;

3) The supernatant was discarded and 100 μl of saline was added to resuspend cells, 2 μl of BCMA flow antibody per tube (ex bioleged cat: 357504 Incubation for 30 minutes at room temperature;

4) Adding proper amount of physiological saline, 800g, centrifuging for 3min, washing twice, and discarding supernatant;

The results show that the cell positive rates of each group are shown in table 3 below:

TABLE 3 TBA Signal transduction receptor expression positive cell occupancy

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The results are shown in Table 3, with a ratio of BCMA positive cells approaching 0 and a ratio of TBA ectodomain positive cells of about 3% in the control cells TIL, indicating that there are substantially no BCMA-expressing positive cells and about 3% of cells expressing a receptor capable of binding TGF-beta 1 (i.e., wild-type TGF-beta receptor) in the prepared native TIL. In TIL-TBA-12-TIL-TBA-17, the positive proportion obtained by the indirect labeling method is higher than that obtained by the direct labeling method, the higher part is probably the wild TGF-beta receptor expressed by TIL itself (which can be considered as false positive in the proportion of the positive cells expressed by the TBA signal conversion receptor), and the proportion of the positive cells obtained by the direct labeling method with the BCMA extracellular domain as a label ranges from about 20% to 50%, and the ratio of the positive TIL expressed by the TBA signal conversion receptor is reflected relatively more accurately.

Since each group of TIL-TBA-1-TIL-TBA-11 cells did not contain BCMA extracellular domain, the ratio of positive cells based on the direct labeling method could not be obtained, and the results of the indirect labeling method were used as the ratio of cells positive for the expression of the TBA signaling receptor for all groups of cells unless otherwise specified in the examples below.

2. Lymphocyte phenotype and cytokine secretion levels of the electrotransport TBA signaling receptor TIL

(1) Lymphocyte phenotype of electrotransport TBA signaling receptor TIL

The associated lymphocyte phenotype of each group of cells is shown in Table 4

TABLE 4 TIL-TBA-1-TIL-TBA-17 cell phenotype

(2) Secretion of the cytokine IFN-gamma for the electrotransport TBA signalling receptor TIL

a. Secretion levels of IFN-gamma without ligand stimulation

The cell supernatants of TIL-TBA-1-TIL-TBA-17 and TIL-CTRL prepared in example 3 were directly taken and assayed for IFN-gamma concentration in the supernatants of each group of cells by the method described in the specification using the CBA assay kit (Human IFN-gamma Flex Set, BDbiosciences, cat# 558269);

b. secretion levels of IFN-gamma following ligand stimulation

Each well of a 12-well plate was charged with the mixture prepared in example 3 and containing 5X 10 ⁵ 1mL of a cell suspension of individual/mL of TIL-TBA-1-TIL-TBA-17 and TIL-CTRL; preparing TGF-beta 1 factor into 5 μg/mL mother liquor with PBS, adding into the above cell suspension to final concentration of 100ng/mL, 37deg.C, 5% CO ₂ The culture was incubated overnight in an incubator, centrifuged, and the supernatant of each sample was collected and assayed for IFN-. Gamma.concentration using a CBA assay kit (Human IFN-. Gamma.Flex Set, BDbiosciences, cat# 558269) according to the protocol described in the specification.

The results are shown in Table 5 below:

TABLE 5 secretion level of TIL-TBA-1-TIL-TBA-17 IFN-gamma

The IFN-gamma secretion level of TIL-TBA-1-TIL-TBA-17 without TGF-beta 1 factor stimulation was not significantly different from that of TIL-CTRL in control cells; after stimulation, the IFN-gamma secretion level of TIL-TBA-1-TIL-TBA-17 is obviously improved compared with that of TIL-CTRL, and the IFN-gamma secretion level of TIL-CTRL is reduced after TGF-beta 1 stimulation compared with that of the TIL-CTRL without stimulation, which is probably caused by the inhibition effect of the added TGF-beta 1 factor on cells after the combination with part of wild TGF-beta receptors on the cell surface in the TIL-CTRL.

The results show that the IFN-gamma secretion level of the TIL which is electrically transformed with the TBA signal conversion receptor is obviously increased after the TIL is stimulated by the TGF-beta 1 factor, which indicates that the TGF-beta 1 factor can stimulate the increase of the activation level of the TIL which expresses the TBA signal conversion receptor.

Example 5: killing effect of overexpression of TBA Signal transduction receptor TIL on homologous tumor cells

The fresh melanoma tissue of example 2 was cut into pieces of 3X 3mm size, and the pieces were mixed as homogeneously as possible and then mixed according to Robert Suriano et al.Ex Vivo Derived Primary Melanoma Cells: implications for Immunotherapeutic Vaccines J Cancer 2013;4 (5) Primary melanoma cells were obtained by culturing according to the method described in section 371-382.Materials and Methods.

The in vitro killing activity of TIL-TBA-1, TIL-TBA-2 … … TIL-TBA-17 and TIL-CTRL cells obtained in example 3 against its cognate melanoma primary cells was examined using a real-time label-free cell function Analyzer (RTCA) from the company Aisen, and the specific procedures were as follows:

(1) Zeroing: adding 50 mu L of DMEM culture solution into each hole, placing into an instrument, selecting the step 1, and zeroing;

(2) Target cell plating: the primary cells of melanoma obtained by culture were cultured at 10 per well ⁴ Spreading individual cells/50 mu L in a plate containing a detection electrode, standing for several minutes, putting the cells into an instrument after the cells are stable, and starting the step 2 to culture the cells;

(3) Adding effector cells: after the target cells are cultured for 18-24 h, observing cell indexes, when the cell indexes are close to 1, respectively adding effector cells TIL-TBA-1, TIL-TBA-2 … … TIL-TBA-17 and TIL-CTRL, wherein the effective target ratio is 2:1 in each hole, starting step 3, and after the co-culture is performed for more than 48h, observing a cell proliferation curve and a killing level, and calculating the target cell killing rate. The target cell killing rate was calculated as follows:

Where A is the cell index of the group to which no effector cells have been added and only target cells (i.e., tumor cells) are present, and B is the cell index of each group to which effector cells have been added.

The results are shown in FIG. 1 and Table 6. FIG. 1 is an exemplary RTCA kill curve for TIL-TBA-13, TIL-TBA-16, and TIL-CTRL against a target cell; table 6 shows the target cell killing rates of TIL-TBA-1-TIL-TBA-17 and TIL-CTRL in RTCA killing assays.

TABLE 6 target cell killing Rate for overexpression of TBA Signal transduction receptor TIL

The results in FIG. 1 show that TIL-TBA-13 and TIL-TBA-16 kill more homologous melanoma primary tumor cells than TIL-CTRL, and that TIL-TBA-16 kills more strongly than TIL-TBA-13. Table 6 shows that TIL-TBA-1-TIL-TBA-17 has significantly higher killing rate for homologous melanoma primary tumor target cells than TIL-CTRL.

Example 6: peripheral blood T cell positive rate of TGF-beta factor stimulated expression TBA signal conversion receptor

PBMC cells overexpressing the TBA signaling receptor were prepared using the expression vectors pKB20-TBA-12, pKB20-TBA-13, pKB20-TBA-16 of example 1 and electro-transferred to PBMC obtained from AllCells, inc. from healthy adult peripheral blood as follows.

1) Collecting the suspension cells into a 50ml centrifuge tube, centrifuging for 3min at 1200 rmp;

2) Discarding the supernatant, adding physiological saline for resuspension, centrifuging for 3min at 1200rmp, discarding physiological saline, and repeating the steps for cell counting;

3) 4 1.5ml centrifuge tubes were taken and 5X 10 tubes were added to each tube ⁶ Individual cells, 1200rmp, were centrifuged for 3min;

4) The supernatant was discarded, and an electrotransfer kit (purchased from Lonza corporation) was used, 18. Mu.L of the solution I reagent and 82. Mu.L of the solution II reagent were added, 5. Mu.g of pKB20 empty plasmid was added to the 1 st tube as a control, 5. Mu.g of pKB20-TBA-12 plasmid was added to the 2 rd tube, 5. Mu.g of pKB20-TBA-13 plasmid was added to the 3 rd tube, and 5. Mu.g of pKB20-TBA-16 plasmid was added to the 4 th tube;

5) Transferring the cell suspension mixed with the plasmid in the centrifuge tube into an electrorotating cup, putting into an electrorotating instrument, selecting a program T020, and performing electric shock;

6) Transferring the electrotransformed cell suspension into a twelve-hole plate (X-VIVO 15 culture solution containing 5% human AB serum) with a micropipette in the kit, mixing, and standing at 37deg.C and 5% CO ₂ Culturing in an incubator; at the same time, 4 wells of the 6-well plate were coated with antibody containing 5. Mu.g/mL OKT-3 and 5. Mu.g/mL CD28, 1mL was added to each well, and the 6-well plate was left to incubate at 37 ℃.

7) After 6 hours, the OKT-3 and CD28 antibody coated well plates were supernatants and the coated wells were washed 3 times with PBS; cells cultured in a 5% CO2 incubator at 37 ℃ after electrotransformation are transferred into a six-hole plate coated with OKT-3 and CD28 antibodies, IL-2 with the final concentration of 100IU/mL is added, culture solution is added until 3mL, after 4-5 days of culture, the growth condition of T cells is observed, and cells expressing signal conversion receptors TBA-12, TBA-13 and TBA-16 and control group cells transferred into pKB20 empty vectors are respectively obtained and are respectively marked as T-TBA-12, T-TBA-13, T-TBA-16 and Mock-T.

TGF-beta factor stimulates T cells expressing TBA signaling receptor

Each well of the 6-well plate was charged with the above-prepared solution containing 1X 10 ⁶ 3mL of each of the cell suspensions of T-TBA-12, T-TBA-13, T-TBA-16 and Mock-T; preparing TGF-beta 1 factor into 5 μg/mL mother liquor with PBS, adding into the above cell suspension to final concentration of 100ng/mL, 37deg.C, 5% CO ₂ And incubating in an incubator for 48 hours, detecting the positive rate by using a flow cytometer, and simultaneously taking the positive rate detected by using the flow cytometer of each cell which is not stimulated to be cultivated by adding the TGF-beta 1 factor under the same condition, and comparing the positive rate with the cells stimulated by the TGF-beta 1 factor. The flow detection method refers to the direct labeling method described in example 4. The results are shown in Table 7:

table 7TGF- β1 factor stimulation to increase TBA expression positive T cell fraction

Compared with the culture condition without TGF-beta 1 factor stimulation, the flow detection result shows that the ratio of the TBA signal conversion receptor expression positive cells of the T-TBA-12, the T-TBA-13 and the T-TBA-16 under the culture condition with the TGF-beta 1 factor stimulation is obviously improved. This indicates that in the presence of the ligand, cells expressing the TBA signaling receptor can be activated and proliferation levels are increased.

Example 7: killing of tumor graft (PDX) tumor tissue by TIL cells expressing TBA Signal transduction receptor

Immunodeficient B-NDG mice (purchased from Baioerskin) were used as PDX model to construct experimental animals.

Experimental design and grouping: as shown in Table 8 below

Table 8 mice dosing regimen and groupings

TILs used in groups 2 to 4 were TIL-CTRL, TIL-TBA-13 and TIL-TBA-16 among the TILs prepared in example 3. Cells were resuspended in PBS by centrifugation prior to tail vein injection to a cell density of 1X 10 ⁸ /mL PBS cell suspension.

Animal feeding

After purchasing the required amount of B-NDG mice, the mice are fed into SPF-class experimental animal houses for 7-10 days.

Environment: the mice will be housed in a clear resin plastic cage in an animal house. The mouse cage padding is the sawdust and corncob padding which are subjected to high-pressure sterilization and is replaced periodically. The animal room is equipped with a high efficiency air filter and the temperature will be maintained between 20-26 c (68-79F) with a relative humidity of 40-70%. Temperature and humidity were continuously observed and recorded. The lighting conditions were 12 hours of fluorescent light illumination and 12 hours of no illumination per day.

Food and drinking water: the experimental mice can obtain special mouse grains (sterilized by irradiation, shanghai Laike laboratory animal liability Co., ltd., china) in an unlimited amount, and can be used for approaching sterilized clean drinking water at any time without obstacle.

Construction of PDX model

1) Patient tumor tissue sample treatment: taking a portion of melanoma tissue from example 2Separating, removing necrotic part tissue, adipose tissue, connective tissue, etc. under aseptic condition, after washing, the tissue is divided into a plurality of 5X 5mm by using a surgical knife ³ The tissue block is placed in a tumor-containing sample transportation preservation solution UW, and B-NDG mice are prepared to be inoculated with the tumor block;

2) Tumor tissue sample inoculation: several B-NDG mice were taken, the mice were fixed with a mouse subcutaneous tumor inoculation fixator after the shoulder blade portion was prepared, the iodophor was sterilized, and the tumor mass in 1) was inoculated to the right inguinal portion with a PDX model tumor mass inoculation trocar after local anesthesia of lidocaine. Inoculation when the astronomical is P0, tumor is measured 2 times per week, and the calculation formula of tumor volume is V=0.5×a×b ² Wherein a and b are the long and short diameters of the tumor, respectively;

3) PDX tissue passaging: observing the growth condition of tumor tissue of each inoculated mouse until the tumor tissue volume is over 300mm ³ After the mice were anesthetized, the tumor mass was removed and cut into 5X 5mm pieces with a scalpel under aseptic conditions ³ Repeating the step 2) after tissue mass, inoculating to the inguinal part of the right side of a new mouse, and waiting for the next generation growth of PDX tumor;

4) Repeating step 3), continuing to subculture for 2-3 generations, taking part of the in-vivo PDX tissue of the mice for histologic pathological analysis, determining that the PDX tissue is still human tissue (but not murine tissue), continuing to inoculate the mice with the PDX tissue (namely, inoculating 48 mice with a PDX tissue block) according to 1.5 times of the number of the mice used in the experimental design of the table 6, observing the tumor formation condition of the mice, measuring the tumor number 2 times per week, and waiting for the tumor formation.

Grouping and administration of animals

Tumor volume of equal PDX inoculated mice reaches 50mm ³ When selecting 32 animals with proper tumor volumes from 48 animals, randomly grouping the animals according to tumor volumes, wherein n=8, and ensuring that all groups have comparability on a base line. Grouping when the diary was D0, dosing was performed according to the protocol of table 6. Animal body weight and tumor volume were measured 3 times a week during the experiment, and animals were observed daily for clinical symptoms. Mm for tumor volume ³ The tumor measurement formula is the same as that described above.

Results

The experimental results are shown in FIG. 2. One-way analysis of variance (one-way ANOVA analysis) was performed on the tumor volume differences between the different groups, followed by post hoc examination (Bonferroni post hoc test) using the Bonferroni method to see if there were significant differences between the different groups. * : p <0.05; * P <0.01. Figure 2 shows that tumor volume increase was very significantly inhibited in the TIL-CTRL dosed mice compared to PBS-injected control tumor-bearing mice by day 40 post-dosing. The tumor volumes of tumor-bearing mice in TIL-TBA-13 and TIL-TBA-16 administration groups were further significantly or very significantly reduced compared to TIL-CTRL administration groups. The result shows that the unmodified TIL-CTRL has obvious inhibition effect on the homologous paired PDX tumor tissues, and the inhibition effect of the TIL on the homologous PDX tumor tissues is improved obviously on the basis after the transgenic modification of the TBA-13 or TBA-16 signal conversion receptor, so that the TBA signal conversion receptor can obviously activate immune effector cells and improve the tumor killing capacity of the immune effector cells. Meanwhile, compared with the TIL-TBA-13 administration group, the tumor volume of the mice in the TIL-TBA-16 administration group on the 40 th day is also obviously smaller, which shows that the TIL-TBA-16 has stronger killing effect on homologous tumor tissues compared with the TIL-TBA-13.

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Numerous modifications and substitutions of details are possible in light of all the teachings disclosed, and such modifications are contemplated as falling within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. An isolated fusion protein comprising: an anti-TGF-beta antibody or antigen-binding fragment or mutant thereof; a transmembrane region or mutant thereof; and an intracellular domain of a costimulatory signaling molecule or a functional fragment or mutant thereof that retains the costimulatory signaling molecule to deliver a costimulatory signal, activate a biological function of an immune cell,

preferably, the anti-TGF-beta antibody is an anti-TGF-beta 1 antibody, an anti-TGF-beta 2 antibody or an anti-TGF-beta 3 antibody.

2. The fusion protein of claim 1, wherein the fusion protein comprises a polypeptide that,

the antibody comprises any one or more selected from single chain antibodies (scFv), single domain antibodies (sdAb), nanobodies (nanobody), heavy chain antibodies (HCAb), fab 'and F (ab') 2, and/or

The costimulatory signaling molecules include one or more of CD28, CD134 (OX 40), CD137 (4-1 BB), LCK, ICOS, DAP, siglec-9, siglec-10, siglec-15, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2R, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21R, CD27, and CD 40; preferably, the costimulatory signaling molecule comprises CD28 and/or IL7Ralpha, and/or

The transmembrane region includes any one or more of the transmembrane regions from CD28, CD134 (OX 40), CD137 (4-1 BB), LCK, ICOS, DAP, siglec-9, siglec-10, siglec-15, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-2R, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21R, CD27, and CD 40; preferably, the IL-2R, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21R is an alpha, beta or gamma subunit, respectively.

3. The fusion protein of claim 1 or 2, wherein the transmembrane region is derived from a CD28 transmembrane region or a mutant thereof or an IL-7Ralpha transmembrane region or a mutant thereof,

preferably, the method comprises the steps of,

the extracellular region of the fusion protein is a TGF-beta single-chain antibody, the transmembrane region is from a CD28 transmembrane region, and the intracellular domain of the costimulatory molecule is a CD28 intracellular domain, or

The extracellular region of the fusion protein is a TGF-beta single-chain antibody, the transmembrane region is from an IL7Ralpha transmembrane region or a mutant thereof, and the intracellular domain of the costimulatory molecule is any one or more selected from a CD28 intracellular region, an IL7Ralpha intracellular region, a CD134 intracellular region, a CD137 intracellular region, an IL-2Rbeta intracellular region, an IL-4Rbeta intracellular region, an IL-7Ralpha intracellular region, an IL-10Ralpha intracellular region, an IL-12Rbeta intracellular region, an IL-15Ralpha intracellular region, an IL-21Ralpha intracellular region, a CD27 intracellular region and a CD40 intracellular region.

4. The fusion protein of claim 1 or 2, wherein the fusion protein comprises a polypeptide of formula (I),

the antibodies have the following HCDR: the HCDR1 amino acid sequence is shown as SEQ ID NO. 44, the HCDR2 amino acid sequence is shown as SEQ ID NO. 45, the HCDR3 amino acid sequence is shown as SEQ ID NO. 46, and/or

The antibodies have the following LCDR: the LCDR1 amino acid sequence is shown as SEQ ID NO. 47, the LCDR2 amino acid sequence is shown as SEQ ID NO. 48, the LCDR3 amino acid sequence is shown as SEQ ID NO. 49, and/or

The amino acid sequence of the CD28 transmembrane region is shown as SEQ ID NO. 6, and/or

The amino acid sequence of the IL-7Ralpha transmembrane region is shown as SEQ ID NO. 12, and/or

The amino acid sequence of the mutant of the IL-7Ralpha transmembrane region is shown in any one of SEQ ID NO 14, 16, 18 and 20, and/or

The amino acid sequence of the CD28 intracellular region is shown as SEQ ID NO. 8, and/or

The amino acid sequence of the IL-7Rα intracellular region is shown in SEQ ID NO. 10, and/or

The amino acid sequence of the CD134 intracellular region is shown as SEQ ID NO. 51,

preferably, the VH amino acid sequence of the antibody is shown as SEQ ID NO. 42, and/or the VL amino acid sequence of the antibody is shown as SEQ ID NO. 43; more preferably, the antibody is a single chain antibody shown in SEQ ID NO. 4.

5. The fusion protein of claim 1 or 2, wherein the signal transduction receptor molecule further comprises a membrane surface tag comprising a BCMA extracellular domain or variant thereof,

preferably, the membrane surface tag further comprises a linker or hinge region located at the N-terminus or C-terminus of the BCMA extracellular domain or variants thereof,

more preferably, the amino acid sequence of the BCMA extracellular domain is shown in SEQ ID No. 22 and/or the amino acid sequence of the linker is shown in SEQ ID No. 24 or 26.

6. A polynucleotide molecule selected from the group consisting of: a polynucleotide molecule or complementary sequence encoding a fusion protein according to any one of claims 1 to 5,

preferably, the method comprises the steps of,

in the fusion protein, the coding sequence of the anti-TGF-beta antibody is shown as SEQ ID NO. 3,

in the fusion protein, the transmembrane region is a CD28 transmembrane region, the coding sequence of the transmembrane region is shown as SEQ ID NO. 5,

in the fusion protein, the transmembrane region is an IL7Ralpha transmembrane region, the coding sequence of the transmembrane region is shown as SEQ ID NO. 11,

in the fusion protein, the intracellular domain of the costimulatory signal molecule is a CD28 intracellular region, the coding sequence of which is shown as SEQ ID NO. 7,

in the fusion protein, the intracellular domain of the costimulatory signal molecule comprises a CD28 intracellular region and a CD134 intracellular region, the coding sequence of the CD28 intracellular region is shown as SEQ ID NO. 7, the coding sequence of the CD134 intracellular region is shown as SEQ ID NO. 50,

In the fusion protein, the intracellular domain of the costimulatory signal molecule is the IL-7Ralpha intracellular region, the coding sequence of which is shown as SEQ ID NO. 9,

in the fusion protein, the intracellular domain of the costimulatory signal molecule is an IL-7Ralpha intracellular region mutant, the coding sequence of which is shown as any one or more of SEQ ID NO 13, 15, 17 and 19,

the fusion protein comprises a membrane surface tag, the membrane surface tag comprises a BCMA extracellular domain with a coding sequence shown as SEQ ID NO. 21,

more preferably, the process is carried out,

the polynucleotide molecule is selected from any one of SEQ ID NO 27-41, 52, 53, or is the complementary sequence of any one of the polynucleotide molecules.

7. A nucleic acid construct comprising the polynucleotide molecule of claim 6,

preferably, the nucleic acid construct is a vector,

more preferably, the vector is an expression vector or an integration vector.

8. A genetically engineered cell expressing the fusion protein according to claim 1 to 5 and/or carrying the coding sequence of the fusion protein,

preferably, the method comprises the steps of,

the cells are immune cells; more preferably, the immune cells include T cells, NK cells, CAR-T, CAR-NK, TCR-T, CIK, NKT and TIL, and/or

The cells also express a CAR, or carry a coding sequence for a CAR, and/or

The cells also express an exogenous TCR, or a coding sequence carrying an exogenous TCR.

9. A pharmaceutical composition comprising any one or more of a pharmaceutically acceptable adjuvant or fusion protein of any one of claims 1-5, a polynucleotide molecule of claim 6, a nucleic acid construct of claim 7, and a genetically engineered cell of claim 8.

10. Use of any one or more of the fusion protein of any one of claims 1-5, the polynucleotide molecule of claim 6, the nucleic acid construct of claim 7, and the genetically engineered cell of claim 8 in the manufacture of a medicament for the treatment or prevention of cancer.