CN116836301B - TGF-beta antibody-based trap receptor - Google Patents

Abstract

The present invention relates to a trap receptor based on TGF-beta antibody, wherein the trap receptor comprises: an anti-TGF-beta antibody or an antigen-binding fragment or mutant thereof as an extracellular region; a transmembrane region or a GPI anchor region. The trap receptor of the present invention can effectively block stimulatory signals that enhance the proliferation of immune cells and reduce the immunosuppressive effect of the TGF-beta factor.

Description

TGF-beta antibody-based trap receptor

Technical Field

The invention relates to the field of biotechnology, in particular to a trap 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- β has a tumor promoting effect, which is generally produced in large amounts in many types of tumors and is known to have carcinogenesis, and which has a negative effect on tumor immunity and significantly inhibits tumor immunity monitoring of the host. TGF- β significantly and directly inhibits the cytotoxic process of Cytotoxic T Lymphocytes (CTLs) by inhibiting transcription of genes encoding a number of key proteins (cytotoxic program), including at least: cytotoxins such as 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.

For negative regulation of immune effector cells by TGF-beta inhibitory signaling pathway, CN110709416A discloses a TGF-beta decoy receptor comprising a TGF-beta binding-region domain and a lipid anchoring region. The TGF-beta binding-region domain comprises a TGF-beta receptor (e.g., TGF-beta RII). The decoy receptor lacks a transmembrane region and an intracellular region, and has no downstream inhibitory signal pathway activation after combining TGF-beta factors, so that the inhibition of TGF-beta on immune effector cells is reduced. In order to stimulate proliferation of immune effector cells and enhance their tumor immunity, there is still a need for more means including work trap receptors to modify 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 as an extracellular region; a transmembrane region or a GPI anchor region or a mutant thereof. The fusion protein does not comprise an intracellular domain or intracellular signaling region of a costimulatory signaling molecule.

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:

HCDR1 comprises the amino acid sequence shown in SEQ ID NO. 48, HCDR2 comprises the amino acid sequence shown in SEQ ID NO. 49, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO. 50.

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

LCDR1 comprises the amino acid sequence shown in SEQ ID NO. 51, LCDR2 comprises the amino acid sequence shown in SEQ ID NO. 52, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO. 53.

In one or more embodiments, the amino acid sequence of the VH of the antibody is shown as SEQ ID NO. 46.

In one or more embodiments, the amino acid sequence of the VL of the antibody is set forth in SEQ ID NO. 47.

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 transmembrane region includes any one or more of the transmembrane regions from CD28、CD134(OX40)、CD137(4-1BB)、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 IL-2R, IL-4R, IL-7R, IL-10R, IL-12R, IL-15R, IL-21R is a respective alpha, beta, or gamma subunit. Preferably, the transmembrane region is the transmembrane region of CD28 or IL7 Ralpha.

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

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

In one or more embodiments, the GPI anchor region comprises one or more proteins selected from the group consisting of or GPI anchor domain ：CD44、CD56、CD73、CD55、Thy1、AchE、IAP、ALPP、CD59、CD14、CD16、CD24、CD28、CD48、CD52、CD58、CD66a、CD66c、CD66d、CD66e、CD67、CD87、CD108、CD157、uPAR、JMH protein, GDNFR, CNTFR, TAG-1, prP, phosphatidylinositol protein, signalin 7, CEA, GFR, ly6G, transferrin receptor, contact element (F3), and T-cadherin. Preferably, the GPI anchor region is a CD52 protein, CD48 protein, CD55 protein, ALPP protein, CD90 protein or a GPI anchor domain thereof.

In one or more embodiments, the amino acid sequence of the GPI anchor domain of the CD52 protein is shown in SEQ ID NO. 12, the amino acid sequence of the GPI anchor domain of the CD48 protein is shown in SEQ ID NO. 14, the amino acid sequence of the GPI anchor domain of the CD55 protein is shown in SEQ ID NO. 16, the amino acid sequence of the GPI anchor domain of the ALPP protein is shown in SEQ ID NO. 18, and the amino acid sequence of the GPI anchor domain of the CD90 protein is shown in SEQ ID NO. 20.

In one or more embodiments, the extracellular region of the fusion protein is a single chain antibody (scFv) to an anti-TGF-beta antibody, which single chain antibody:

(1) Has the following characteristics CDR：HCDR1：SEQ ID NO:48,HCDR2：SEQ ID NO:49,HCDR3：SEQ ID NO:50,LCDR1：SEQ ID NO:51,LCDR2：SEQ ID NO:52,LCDR3：SEQ ID NO:53,

(2) Has the following variable regions: VH: SEQ ID NO. 46, VL: SEQ ID NO. 47, or

(3) As shown in SEQ ID NO. 4,

And

The transmembrane region includes any one or more of the transmembrane regions from CD28、CD134(OX40)、CD137(4-1BB)、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 CD40,

The GPI anchor region comprises one or more proteins selected from the group consisting of the following or GPI anchor domain ：CD44、CD56、CD73、CD55、Thy1、AchE、IAP、ALPP、CD59、CD14、CD16、CD24、CD28、CD48、CD52、CD58、CD66a、CD66c、CD66d、CD66e、CD67、CD87、CD108、CD157、uPAR、JMH proteins, GDNFR, CNTFR, TAG-1, prP, phosphatidylinositol protein, signalin 7, CEA, GFR, ly G, transferrin receptor, contact element (F3), and T-cadherin.

In one or more embodiments, the fusion protein comprises a single chain antibody as set forth in SEQ ID NO. 4, and a GPI anchor domain of CD28 as set forth in SEQ ID NO. 8, IL7Ralpha as set forth in SEQ ID NO. 10, or CD52 as set forth in SEQ ID NO. 12.

In one or more embodiments, the fusion protein further includes a membrane surface tag located at the N-terminus or C-terminus of the anti-TGF-beta antibody or antigen-binding fragment or mutant thereof. Preferably, the membrane surface tag comprises a BCMA extracellular domain or variant thereof. Preferably, the amino acid sequence of the BCMA extracellular domain is shown in SEQ ID NO. 22.

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.

In one or more embodiments, the fusion protein further comprises a signal peptide. Preferably, the signal peptide is a CD8 signal peptide, TBRII signal peptide or a CD52 signal peptide.

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 in the fusion protein is a CD28 transmembrane region having the coding sequence shown in SEQ ID NO. 7.

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. 9.

In one or more embodiments, the GPI anchor region in the fusion protein is the GPI anchor domain of CD52, which has the coding sequence shown in SEQ ID NO. 11.

In one or more embodiments, the GPI anchor region in the fusion protein is the GPI anchor domain of CD48, which has the coding sequence shown in SEQ ID NO. 13.

In one or more embodiments, the GPI anchor region in the fusion protein is the GPI anchor domain of CD55, which has the coding sequence shown in SEQ ID NO. 15.

In one or more embodiments, the fusion protein wherein the GPI anchor region is the GPI anchor domain of ALPP, has the coding sequence shown in SEQ ID NO. 17.

In one or more embodiments, the GPI anchor region in the fusion protein is the GPI anchor domain of CD90, which has the coding sequence shown in SEQ ID NO. 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.31-45 and SEQ ID NOS.54-55, 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; including viral vectors or non-viral vectors, preferably non-viral vectors.

In one or more embodiments, the vector is an expression vector or an integration 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, DN 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 method of sorting cells expressing the fusion protein comprising detecting the anti-TGF-beta antibody or antigen binding fragment or mutant thereof or the membrane surface tag on the cell surface.

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.

The invention has the advantages that: compared with the conventional TGF-beta trap receptor, the trap receptor based on the TGF-beta antibody has higher affinity with TGF-beta factor, can better capture the TGF-beta factor secreted by tumor cells, reduce the inhibition effect suffered by immune effector cells, and improve the activation level of the immune effector cells; and the transmembrane region adopts a GPI protein membrane surface anchoring sequence, so that the effect can be further improved through the aggregation effect brought by the lipid raft.

Drawings

Fig. 1: in vivo killing effect of TIL-CTRL, TIL-TBDR-7 and TIL-TBDR-15 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.

Compared with the trap receptor disclosed in CN110709416A and based on the extracellular region of TGF-beta receptor, the anti-TGF-beta antibody is used as the extracellular region of the trap receptor, has higher affinity with TGF-beta factor, can better reduce the immunosuppressive effect of TGF-beta factor, and further improves the activation and proliferation level of immune effector cells expressing the trap 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 trap receptors of the invention and/or contain coding sequences for the trap receptors. The trap receptors of the present invention are designed to bind inhibitory molecules but no intracellular region triggers a downstream inhibitory signal pathway. That is, the ligand triggering the inhibitory signal is occupied, reducing the inhibition of immune effector cells by TGF-beta factors.

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., trap 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 "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'sPharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995), and include, but are not limited to, pH modifiers, surfactants, adjuvants, ionic strength enhancers, e.g., pH modifiers include, but are not limited to, phosphate buffers, surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, e.g., 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.

Trap receptors

The trap receptor of the present invention is a fusion protein comprising a TGF-beta antibody or antigen binding fragment thereof fused to a transmembrane region or GPI anchor. More specifically, the trap receptors of the present invention include TGF-beta antibodies or antigen binding fragments thereof as extracellular regions and transmembrane or GPI-anchor regions. The trap receptor does not comprise an intracellular domain or intracellular signaling region of a costimulatory signaling molecule. Further, the trap receptor does not comprise an intracellular region.

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. The F (ab ') ₂ antibody fragment was originally generated as a pair 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. 48. In certain embodiments, the HCDR2 of an anti-TGF-beta antibody described herein is set forth in SEQ ID NO. 49. In certain embodiments, the HCDR3 of the anti-TGF-beta antibodies described herein is set forth in SEQ ID NO. 50. In certain embodiments, the LCDR1 of the anti-TGF-beta antibodies described herein is set forth in SEQ ID NO. 51. In certain embodiments, the LCDR2 of the anti-TGF-beta antibodies described herein is set forth in SEQ ID NO: 52. In certain embodiments, the LCDR3 of the anti-TGF-beta antibodies described herein is set forth in SEQ ID NO. 53. In certain embodiments, the VH of the anti-TGF-beta antibodies described herein is as set forth in SEQ ID NO. 46. In certain embodiments, the VL of an anti-TGF-beta antibody described herein is set forth in SEQ ID NO. 47.

In the present invention, an anti-TGF-beta antibody is localized on the outer surface of a cell membrane by a transmembrane region or GPI anchor. The transmembrane region or GPI anchor region is located at the C-terminus of the anti-TGF-beta antibody.

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 CD28、CD134(OX40)、CD137(4-1BB)、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 CD40 or mutants thereof that retain transmembrane function. In the present invention, the preferred transmembrane region is from the CD28 or IL-7Ralpha transmembrane region. Exemplary amino acid sequences and coding sequences for the CD28 transmembrane region are shown in SEQ ID NOS.8 and 7, respectively. Exemplary amino acid sequences and coding sequences for the IL-7Ralpha transmembrane region are shown in SEQ ID NOS 10 and 9, respectively. 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.

GPI anchor regions include one or more selected from the group consisting of GPI anchor domains (also referred to herein as GPI signal sequence )：CD44、CD56、CD73、CD55、Thy1、AchE、IAP、ALPP、CD59、CD14、CD16、CD24、CD28、CD48、CD52、CD58、CD66a、CD66c、CD66d、CD66e、CD67、CD87、CD108、CD157、uPAR、JMH protein, GDNFR, CNTFR, TAG-1, prP, phosphatidylinositol protein, signalin 7, CEA, GFR, ly G, transferrin receptor, contact element (F3) and T-cadherin; alternatively, the GPI anchor regions are CD52 protein, CD48 protein, CD55 protein, ALPP protein, CD90 protein or GPI anchor domains thereof. GPI anchor proteins or anchor domain sequences thereof are known in the art. Furthermore, the anchor domain sequences thereof can be readily obtained by those skilled in the art based on the sequence of GPI anchor proteins).

In one or more embodiments, the GPI anchor region has the sequence set forth in any one of SEQ ID NOs 12, 14, 16, 18, 20, or a variant thereof having at least 90% sequence identity thereto and having GPI-binding functionality.

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 extracellular region as described herein refers to a fragment in the complete sequence that retains the biological function of the structure formed by the complete sequence of the extracellular region, e.g., binding to a corresponding ligand, antigen, etc., and a functional fragment of each extracellular domain, as well as a functional fragment of each transmembrane region or GPI anchor, suitable for use in the present invention can be readily determined by one skilled in the art in combination with means of the prior art.

The trap receptors 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 extracellular domain comprises the amino acid sequence shown in SEQ ID No. 22; the coding nucleic acid sequence comprises a sequence shown as SEQ ID NO. 21. The membrane surface tag may function as an immunobraking element, recognition element, linker, element that induces ADCC, ADCP and/or CDC effects, and may also function as a sorting tag for cells positive for expression of the trap receptor.

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 26; or the BCMA extracellular domain is directly linked to the N-terminus of the anti-TGF-beta antibody shown in SEQ ID NO. 4.

The "mutant" as referred to herein includes mutants of each antibody, transmembrane region, GPI anchor and membrane surface tag, as long as the mutants retain the respective biological functions of the antibody, transmembrane region, GPI anchor and membrane surface tag. 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 GPI anchor regions 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 GPI anchor region as a comparison; mutants suitable for use in the membrane surface tags of 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 membrane surface tag as a comparison. Or 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 residue insertions, substitutions or deletions 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 aforementioned trap receptors, 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 trap receptor. More specifically, the 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 trap receptors described above. Such mutants retain the biological function of the trap receptors of the present invention, including but not limited to blocking signals caused by TGF-beta that inhibit immune cell proliferation. Mutations may occur in any, any two, or all three of the extracellular domains, transmembrane regions, and GPI anchor regions 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.

Exemplary trap receptors of the present invention include, but are not limited to, trap receptors comprising or consisting of an extracellular domain, transmembrane region or GPI anchor as shown in each row of Table 1 below:

TABLE 1

Extracellular domain	Transmembrane region or GPI anchoring region
		TGF-βscFv	CD28 transmembrane region
BCMA extracellular domain-TGF-beta scFv	CD28 transmembrane region
		TGF-βscFv	IL7Ralpha transmembrane region
TGF-βscFv	CD52 GPI anchor domain
		TGF-beta scFv-linker 1-BCMA extracellular domain	CD52 GPI anchor domain
TGF-beta scFv-linker 2-BCMA extracellular domain	CD52 GPI anchor domain
		BCMA extracellular domain-TGF-beta scFv	CD52 GPI anchor domain
TGF-βscFv	CD48 GPI anchor domain
		TGF-beta scFv-linker 1-BCMA extracellular domain	CD48 GPI anchor domain
TGF-beta scFv-linker 2-BCMA extracellular domain	CD48 GPI anchor domain
		BCMA extracellular domain-TGF-beta scFv	CD48 GPI anchor domain
TGF-βscFv	CD55 GPI anchor domain
		TGF-beta scFv-linker 1-BCMA extracellular domain	CD55 GPI anchor domain
TGF-beta scFv-linker 2-BCMA extracellular domain	CD55 GPI anchor domain
		BCMA extracellular domain-TGF-beta scFv	CD55 GPI anchor domain
TGF-βscFv	ALPP GPI anchor domain
		TGF-beta scFv-linker 1-BCMA extracellular domain	ALPP GPI anchor domain
TGF-beta scFv-linker 2-BCMA extracellular domain	ALPP GPI anchor domain
		BCMA extracellular domain-TGF-beta scFv	ALPP GPI anchor domain
TGF-βscFv	CD90 GPI anchor domain
		TGF-beta scFv-linker 1-BCMA extracellular domain	CD90 GPI anchor domain
TGF-beta scFv-linker 2-BCMA extracellular domain	CD90 GPI anchor domain
		BCMA extracellular domain-TGF-beta scFv	CD90 GPI anchor domain
TGF-beta scFv-linker 1-BCMA extracellular domain	CD28 transmembrane region

In some embodiments, the trap receptors described herein further comprise a signal peptide. Preferably, the signal peptide is located at the N-terminus of the trap 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 the signal peptide of CD8, CD4, CD28, CD52, CD137, EGFR, TGFBRI, TGFBRII (TBRII), TGFBRIII or the light chain signal peptide. In some embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO. 2, 6 or 28, the coding sequences of which are set forth in SEQ ID NO. 1, 5, 27, respectively.

It will be appreciated that linkages between the extracellular domains described herein and the transmembrane or GPI anchor, between the antibody and the membrane surface tag, between the signal peptide and other extracellular domains may be made by linker sequences, 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.

In some embodiments, the fusion proteins described herein comprise any one of the following: (a) a TGF- βscfv and CD28 transmembrane region having 6 CDRs as described herein, (b) a BCMA extracellular domain, a TGF- βscfv and CD28 transmembrane region having 6 CDRs as described herein, (c) a TGF- βscfv and IL7 ra transmembrane region having 6 CDRs as described herein, (d) a GPI anchor domain having a TGF- βscfv and CD52 or CD48 or CD55 or ALPP or CD90 having 6 CDRs as described herein, (e) a TGF- βscfv having 6 CDRs as described herein, linker 1, BCMA extracellular domain, GPI anchor domain of CD52, (f) a TGF- βscfv having 6 CDRs as described herein, linker 2, BCMA extracellular domain, GPI anchor domain of CD52, (g) a TGF- βscfv and CD52 GPI anchor domain having 6 CDRs as described herein, (h) a TGF- βscfv having 6 CDRs as described herein, BCMA extracellular domain, linker 1 and CD28, wherein the transmembrane domain of BCMA has the amino acid sequence shown in SEQ ID No. 2, and the transmembrane domain of SEQ ID No. 2 is shown in SEQ ID No. 2.

In one or more embodiments, the fusion protein comprises any one of the following: (aa) a CD8 signal peptide, a TGF- βscFv and CD28 transmembrane region having 6 CDRs described herein, (bb) a CD8 signal peptide, a TGF- βscFv and IL7Rα transmembrane region having 6 CDRs described herein, (cc) a TBRII signal peptide, a GPI-anchor domain of a TGF- βscFv and CD52 having 6 CDRs described herein, (dd) a TBRII signal peptide, a TGF- βscFv having 6 CDRs described herein, a linker 1, a BCMA extracellular domain, a GPI-anchor domain of CD52, (ee) TBRII signal peptide, a TGF- βscFv having 6 CDRs described herein, a linker 2, a BCMA extracellular domain, a GPI-anchor domain of CD52, (ff) a CD52 signal peptide, a TGF- βscFv having 6 CDRs described herein, a GPI-anchor domain of CD52, (gg) a CD52 signal peptide, a TGF- βscfv having 6 CDRs as described herein, linker 1, BCMA extracellular domain, a GPI-anchored domain of CD52, (hh) a CD52 signal peptide, a TGF- βscfv having 6 CDRs as described herein, linker 2, BCMA extracellular domain, a GPI-anchored domain of CD52, (ii) a CD52 signal peptide, linker 1, BCMA extracellular domain, a GPI-anchored domain of TGF- βscfv having 6 CDRs as described herein and CD52, (jj) a CD52 signal peptide, linker 2, BCMA extracellular domain, a GPI-anchored domain of TGF- βscfv having 6 CDRs as described herein and CD52, (kk) a TBRII signal peptide, a GPI-anchored domain of TGF- βscfv having 6 CDRs as described herein and CD52, linker 2, BCMA extracellular domain, and CD28 transmembrane region, wherein the CD8 signal peptide is as set forth in SEQ ID NO:2, TBRII signal peptide is shown as SEQ ID NO. 6, the CD52 signal peptide is shown as SEQ ID NO. 28, the CD28 transmembrane region is shown as SEQ ID NO. 8, the IL7Rα transmembrane region is shown as SEQ ID NO. 10, the GPI anchoring domain of CD52 is shown as SEQ ID NO. 12, the BCMA extracellular domain is shown as SEQ ID NO. 22, the linker 1 is shown as SEQ ID NO. 24, and the linker 2 is shown as SEQ ID NO. 26.

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 (Flavin reductase), PSMA (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 mucin 1), CD22, CD23, CD30, CD33, CD44v7/8, CD70, VEGFR1, VEGFR2, IL-11R/, EGP-2, EGP-40, FBP, GD3 (also known as ST8SIA 1), PSCA (prostate stem cell antigen), FSA (also known as KIAA 1109), PSA (also known as KLK 3), HMGA2, fetal acetylcholine receptor, leY (also known as FUT 3), epCAM, MSLN (mesothelin), IGFR1, EGFR, EGFRvIII, ERBB, ERBB4, CA125 (also known as MUC 16), CA15-3, CA19-9, CA72-4, CA242, CA50, CYFRA21-1, SCC (also known as SERPINB 3), AFU (also known as flu 1), VCA, pov-64 and oxyntopin (grep).

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 the 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, T a 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 (Flavin reductase), PSMA (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 (mucin 1), CD22, CD23, CD30, CD33, CD44v7/8, CD70, VEGFR1, VEGFR2, IL-11R/, EGP-2, EGP-40, FBP, GD3 (aka ST8SIA 1), PSCA (prostate stem cell antigen), FSA (aka KIAA 1109), PSA (aka KLK 3), HMGA2, fetal acetylcholine receptor, leY (aka FUT 3), epCAM, MSLN (mesothelin), IGFR1, EGFR, EGFRvIII, ERBB3, ERBB4, CA125 (aka MUC16, mucin 16), CA15-3, CA19-9, CA72-4, CA242, CA50, cya 21-1, afhenb 3), afpib 1, AFP (aka 53), fuv (aka) and oxyntopin (pov) gastric secretion proteins (pov) and (pov 64).

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 trap receptors of the present invention. The invention also provides the complement of the coding sequence of the trap 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 the trap receptor of the present invention, which is a nucleic acid construct comprising a promoter, a trap 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 prince, 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 trap receptor and the CAR of the invention. The vector may be a bicistronic. The coding sequence of the CAR may be disposed 5 'or 3' to the trap receptor coding sequence. Expression of the CAR and trap 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 (2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory), ausubel et al (1989,Short Protocols in Molecular Biology,Wiley), or other techniques described in standard textbooks. 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 et al (supra).

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 trap receptors of the invention and/or the coding sequences thereof. 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 trap receptor modified cells 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 trap receptors and/or their coding sequences of the present invention, the cells of the present invention may have one or more other 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, αβ T 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 trap receptors as described herein) may be administered parenterally, e.g., intravenously, intraarterially, intrathecally, subdermally, 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 vehicles 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 trap receptor, the polynucleotide molecule, the vector, the host cell and the pharmaceutical composition containing the trap receptor, the polynucleotide molecule, the vector and the host cell 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 trap 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 trap 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 trap 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 the trap receptor of any of the embodiments of the present invention and/or reagents for generating one or more cells for use in cell therapy comprising a recombinant expression vector may be included in a kit. 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.

The components of the kit may also be provided in the form of a cryopreservation formulation (e.g., a cryopreservation solution). The frozen preparation can be directly infused into a patient or formulated as an infusion composition after thawing. Thus, the kit may also comprise a cell cryopreservation bag, a cell cryopreservation tube, a temperature holding means (e.g. a container comprising liquid nitrogen), a thawing means, etc.

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 trap receptors 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 identified 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, ind. Molecular cloning Experimental guidelines, third edition, scientific Press, et al), corresponding references, or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Examples

Example 1: construction of trap 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 described in this example, a pKB20 vector containing an expression cassette for a foreign gene was constructed. Specifically, the sequences shown in SEQ ID NOS.31-45 and SEQ ID NOS.54-55, representing different TGF- β trap receptors (TBDR), were obtained synthetically by the entrusting company, as shown in Table 2 below. The 2-terminal of the sequences shown in SEQ ID Nos. 31 to 45 and 54 to 55 were ligated with a linker containing the 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, designated pKB20-TBDR-1, pKB20-TBDR-2, and pKB20-TBDR-3 … … pKB20-TBDR-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.

Table 2: TGF-beta trap receptor structure and sequence

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) The adipose tissue and necrotic tissue were removed with a sterile scalpel, the tumor tissue was cut into small pieces of 3X 3mm ³ in diameter, 2G-REX 100 culture pots (from Wilsonwolf) were taken, 42 randomly selected tumor tissue blocks are placed in each G-REX100 culture tank, a seed cell culture medium is added into the culture tank, and the seed culture medium comprises the following components: 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 CryoStor10 (purchased from BioLifeSolutions) frozen liquid 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, culturing the tumor tissue blocks at 37 ℃ with 5% CO ₂, removing half of the old seed cell culture medium every 4 days, supplementing half of the fresh seed cell culture medium, and counting the total number and the activity rate of TIL seed cells after centrifugation is carried out on 12 th day;

5) The seed cells harvested in 4) were resuspended to 5.0X10 ⁵/mL with an expansion 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, added to a cell culture vessel pretreated with anti-CD 3 mAb, anti-CD 28 mAb and anti-CD 137 mAb coating, activated for 2 days at 37℃with 5% CO ₂, and the activated cells were collected by centrifugation and inoculated into a G-REX500M culture tank to which the expansion medium having been preheated beforehand was added, and the expansion medium in the G-REX500M culture tank was identical to the expansion medium described above. The volume of the expansion medium in each G-REX500M was 5L. The activated seed cells are inoculated according to the inoculation density of 2.5 multiplied by 10 ⁵/cm², are cultured at 37 ℃ by 5 percent CO ₂, after cell counting is carried out every 4 days, half volume of old expansion culture medium is removed, half volume of fresh expansion culture medium is added, after the total number of cells in each G-REX500M tank reaches 1.0 multiplied by 10 ¹⁰, the seed cells are split according to the proportion of 1:2, and after each seed cell is added with the fresh expansion culture medium to 5L, the seed cells are continuously cultured. 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 where 5% CO ₂ at 37℃was pre-heated for 1 hour;

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

	100μL NucleocuvetteTMStrip(μL)
		volume of Nucleofector TM solution	82
Electrolysis supplementary solution	18

The plasmids pKB20-TBDR-1, pKB20-TBDR-2, pKB20-TBDR-3 … … pKB20-TBDR-17 and the control empty plasmid pKB20 tested as required, an experimental group electrotransport system 17 and a control group 1 were prepared;

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

4) Adding plasmids pKB20-TBDR-1, pKB20-TBDR-2, pKB20-TBDR-3 … … pKB20-TBDR-17 and control empty plasmid pKB20 6 mug into the electrotransfer solution of each of the different experimental groups and the control groups prepared in 2), and standing at room temperature for less than 30 min;

5) Resuspending all test tubes with the plasmid-containing electrotransfer solution prepared in 4), 100 μl of each tube, carefully sucking the cell resuspension into a LONZA 100 μl electrotransfer cup, placing the electrotransfer cup into LONZA Nucleofector ^TM b electrotransfer tank, starting electrotransfer program, and selecting X001;

6) Carefully taking out the electric rotating cup after the electric rotating, sucking the cell suspension, transferring the cell suspension into an EP tube, adding 200 mu L of preheated AIM-V culture medium into each tube, and then transferring the cell suspension into a hole containing the preheated AIM-V culture medium in a 12-well plate in 1), and culturing at 37 ℃ with 5% CO ₂; after 5 days of culture, TIL cells overexpressing TBDR-1-TBDR-17 and control TIL cells, designated TIL-TBDR-1, TIL-TBDR-2 … … TIL-TBDR-17 and TIL-CTRL, respectively, were obtained.

Example 4: phenotypic assay of TBDR-expressing TIL

1. Cell viability and positivity of TIL of electrotransport TBDR

Cell viability was measured for each group by trypan blue staining and cell counter counting. The results show that TIL-TBDR-1-TIL-TBDR-17 and TIL-CTRL prepared in example 3 have cell viability above 95%.

The TIL of each electrotransport TBDR was fluorescently labeled by an indirect labeling method, and the flow cytometer detected the cell fraction positive for TBDR receptor gene expression, as follows:

1) Collecting TIL-TBDR-1-TIL-TBDR-17 and TIL-CTRL groups of cells, collecting 1× ⁶ cells per group, 800g, and 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 conjugated biotin was added to each tube at a concentration of 1. Mu.g/mL cytokine TGF-. Beta.1 (purchased from acrobiosystems; cargo number: TG 1-H8217), 30min incubation at room temperature; 800g, centrifuging for 3min, and washing twice;

4) The supernatant was discarded, 100 μl of saline was added per tube of pellet, cells were resuspended, and 2 μl of PE-streptavidin (PE STREPTAVIDIN, available from BDbiosciences; cargo number: 554061 30min, 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, for TIL (TIL-TBDR-4, TIL-TBDR-5, TIL-TBDR-7, TIL-TBDR-8, TIL-TBDR-9, TIL-TBDR-10, TIL-TBDR-16, TIL-TBDR-17) expressing TBDR receptor containing BCMA extracellular domain, the ratio of BCMA-positive cells was detected by direct labeling flow cytometry using BCMA extracellular domain (or mutant thereof) as a tag, as follows:

1) Collecting cells of TIL-TBDR-4, TIL-TBDR-5, TIL-TBDR-7, TIL-TBDR-8, TIL-TBDR-9, TIL-TBDR-10, TIL-TBDR-16 and TIL-TBDR-17, collecting 1×10 ⁶ cells per cell, 800g, and 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;

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

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

TABLE 3 Indirect labelling and direct labelling detection TBDR Duty ratio of positive cells expressed

The results are shown in Table 3, in the control cells TIL-CTRL, the ratio of BCMA positive cells was 0 and the ratio of cells expressing the TGF- β receptor was about 17%, indicating that in the prepared native TIL, there were no BCMA-positive cells and about 17% of cells expressed the native TGF- β receptor. In TIL-TBDR-4, TIL-TBDR-5, TIL-TBDR-7, TIL-TBDR-8, TIL-TBDR-9, TIL-TBDR-10, TIL-TBDR-16, and TIL-TBDR-17, the positive proportion obtained by the indirect labeling assay was higher than that obtained by the direct labeling assay, the higher proportion was substantially identical to that of TIL of the native TGF-beta receptor expressed by itself (which could be considered as a false positive part in the indirect result), whereas the proportion of positive cells obtained by the direct labeling method with BCMA extracellular domain as a tag ranged from about 50% to 60%, reflecting the proportion of positive TIL expressed by TBDR relatively accurately.

The following examples relate to the results of the positivity rates of TIL-TBDR-4, TIL-TBDR-5, TIL-TBDR-7, TIL-TBDR-8, TIL-TBDR-9, TIL-TBDR-10, TIL-TBDR-16 and TIL-TBDR-17 using direct labeling, TIL-TBDR-1, TIL-TBDR-2, TIL-TBDR-3, TIL-TBDR-6, TIL-TBDR-11, TIL-TBDR-12, TIL-TBDR-13, TIL-TBDR-14 and TIL-TBDR-15 using indirect labeling minus the TIL-CTRL indirect labeling positive rate value (17.2%).

2. Lymphocyte phenotype of TIL of electrotransport TBDR

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

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

Sample name	CD45+(％)	CD3+(％)	CD4+(％)	CD8+(％)
					TIL-TBDR-1	100	99.52	41.66	54.89
TIL-TBDR-2	100	96.21	36.75	54.62
					TIL-TBDR-3	99.97	96.75	32.21	57.13
TIL-TBDR-4	100	99.27	24.98	54.78
					TIL-TBDR-5	99.89	94.17	31.66	59.43
TIL-TBDR-6	99.95	96.76	42.18	51.08
					TIL-TBDR-7	100	92.24	29.46	54.04
TIL-TBDR-8	100	98.08	42.01	52.86
					TIL-TBDR-9	99.99	94.85	17.89	65.45
TIL-TBDR-10	99.86	91.97	32.05	53.88
					TIL-TBDR-11	100	90.01	37.14	52.21
TIL-TBDR-12	99.94	92.97	24.87	61.01
					TIL-TBDR-13	100	94.81	30.09	61.98
TIL-TBDR-14	100	97.44	42.16	50.72
					TIL-TBDR-15	99.98	99.65	35.97	60.71
TIL-TBDR-16	100	93.86	29.14	62.16
					TIL-TBDR-17	99.79	90.25	40.77	47.85
TIL-CTRL	100	96.59	41.39	52.91

Example 5: inhibition of TGF-beta mediated signaling in TIL expressing TBDR

After incubating TIL-TBDR-1-TIL-TBDR-17 and TIL-CTRL obtained in example 3 with a final concentration of 50ng/mL TGF- β1 factor at 37℃under 5% CO ₂ for 1 hour, the proportion of cells phosphorylated at the intracellular downstream signaling pathway of the TGF- β receptor in each sample was examined using the BD ^TM Phosphoflow protocol of BD Biosciences as follows:

1) The fixation buffer was heated in a 37 ℃ water bath 5-10 minutes prior to use (BD ^TM Phosflow Fix Buffer I, cat No.:557870,BD Biosciences,);

2) Centrifuging 300g of 5min to collect TIL-TBDR-1-TIL-TBDR-17 and TIL-CTRL treated by TGF-beta 1, immediately fixing the cells with a fixing buffer to maintain the phosphorylation state of the cells, and mixing the cells by inverting or swirling an EP tube;

3) Incubating the cells at 37℃for 10-15 minutes;

4) Centrifuging at 300g for 5 minutes to remove the supernatant;

5) Adding a permeation buffer (BD ^TMPhosflow Perm Buffer III,Cat.No.：558050,BD Biosciences,1×10⁶ cells/mL), standing at room temperature, and incubating for 10 minutes;

6) Washing the cells twice with a permeabilizing buffer; centrifuging at 300g for 5 minutes to remove the supernatant;

7) Resuspend cells at a density of 1X 10 ⁷ cells/mL with permeabilized buffer;

8) 1X 10 ⁶ cells were taken and 5. Mu.L of Smad2/3 fluorescent antibody (BD Phosflow ^TM Alexa 647Anti-Smad2 (pS 465/pS 467)/Smad 3 (pS 423/pS 425), cat.No.:562696,BD Biosciences), incubated for 30 minutes at room temperature protected from light;

9) 2mL of staining Buffer (BD Pharmingen ^TM Stain Buffer (FBS), cat. No.:554656,BD Biosciences) washing once; centrifuging at 300g for 5 minutes, and removing the supernatant;

10 Resuspension with 500 μl of staining buffer, detection by up-flow cytometry.

The proportion of phosphorylated Smad2/3 positive cells (Smad 2/3P%) in each sample (TIL-TBDR-1-TIL-TBDR-17) obtained by flow assay was homogenized according to the TBDR expression positive rate (see example 4) to obtain homogenized Smad2/3P% (NSmad 2/3P%), smad2/3P% of TIL-CTRL was the control Smad2/3P%, and the inhibition rate of TGF-beta signaling in each sample cell was calculated as follows:

The results are shown in Table 5:

Table 5: TGF-beta signaling inhibition in TIL-TBDR-1-TIL-TBDR-17

The results in Table 5 show that TBDR (TBDR-3-TBDR-10), which structurally has a TGF-beta scFv extracellular region and GPI anchor region, significantly inhibits TGF-beta signaling over TBDR of the other structure. TBDR possessing GPI anchor region also inhibited TGF- β signaling significantly more than the structure whose transmembrane region was the conventional transmembrane region (CD 28 or IL7Rα transmembrane region) (TBDR-17 vs TBDR-1\TBDR-2). In addition, of the several TBDR containing different GPI anchor regions (TBDR-11-TBDR-15), TBDR (TBDR-11\16) with the CD52 GPI anchor region resulted in the highest inhibition of TGF- β signaling and was significantly higher than TBDR (TBDR-12\13\14\15) containing other GPI anchor regions.

Example 6: killing effect of TIL overexpressing TBDR 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 uniformly as possible and cultured according to the method described in section Robert Suriano et al.Ex Vivo Derived Primary Melanoma Cells:Implications for Immunotherapeutic Vaccines J Cancer 2013;4(5):371-382.Materials and Methods to obtain primary melanoma cells. After tumor primary cells had adhered, the supernatant was taken and assayed for TGF- β1 concentration according to the instructions of the TGF- β1ELISA kit (Human TGF beta 1ELISA Kit,Cat.No: ab100647, abcam). The results showed that the concentration of TGF- β1 in the culture supernatant was 12.7ng/mL.

The in vitro killing activity of TIL-TBDR-1, TIL-TBDR-2 … … TIL-TBDR-17 and TIL-CTRL cells obtained in example 3 on their cognate melanoma primary cells was examined using a real-time label-free cell function Analyzer (RTCA) from the company Aisen, and the specific steps 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: paving 10 ⁴ cells per 50 mu L of melanoma primary cells obtained by culture in a plate containing a detection electrode, standing for a plurality of minutes, putting the cells into an instrument after the cells are stabilized, and starting the step 2 to culture the cells;

(3) Adding effector cells: after the target cells are cultured for 18-24 h, observing the cell index, when the cell index is 1, respectively adding effector cells TIL-TBDR-1, TIL-TBDR-2 … … TIL-TBDR-17 and TIL-CTRL, wherein the effective target ratio is 2:1 in each hole, starting the step 3, after the co-culture is more than 48, observing the cell proliferation curve, and calculating the killing rate of the target cells. 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 Table 6. Compared with TIL-CTRL, TIL-TBDR-1 and TIL-TBDR-2 … … TIL-TBDR-17 have obviously stronger killing effect on homologous melanoma primary tumor cells. Meanwhile, compared with the killing of TIL-TBDR-11-TIL-TBDR-16 on tumor cells, the killing power of TIL-TBDR-1-10 is obviously stronger. Compared with a trap receptor based on a TGF-beta receptor II extracellular domain, the trap receptor based on TGF-beta scFv can better reduce the immunosuppressive effect of TGF-beta factors, and promote the activation level of immune effector cells, thereby further promoting the killing of tumor cells. And compared with the conventional transmembrane region, the function of a trap receptor can be further improved through TBDR anchored on the surface of the membrane by the GPI anchoring region, and the killing effect on target cells is improved.

Table 6: kill Rate of TIL expressing TBDR on homologous target cells

Example 7: killing of tumor graft (PDX) tumor tissue by TIL cells expressing TBDR

Experimental animal

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

Experimental design and grouping: as shown in Table 7 below

Table 7: mouse dosing regimen and grouping

TILs used in groups 2 to 4 were TIL-CTRL, TIL-TBDR-7 and TIL-TBDR-15 among the TILs prepared in example 3. Cells were resuspended in PBS by centrifugation prior to tail vein injection to give a cell suspension with a cell density of 1X 10 ⁸/mL PBS.

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 part of melanoma tissues in the example 2, removing necrotic tissues, adipose tissues, connective tissues and the like under aseptic conditions, dividing the tissue into a plurality of tissue blocks of 5X 5mm ³ by using a scalpel after washing, placing the tissue blocks in a tumor-containing sample transportation and preservation solution UW, and preparing to inoculate B-NDG mice with the tumor blocks;

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. Inoculating when the astronomical is P0, measuring tumor 2 times per week, wherein the calculation formula of tumor volume is V=0.5×a×b ², and a and b are the long diameter and short diameter of tumor respectively;

3) PDX tissue passaging: observing the growth condition of tumor tissues of each inoculated mouse, after the tumor tissue volume is over 300mm ³, anaesthetizing the mouse, taking out tumor blocks, cutting the tumor blocks into 5X 5mm ³ tissue blocks by a scalpel under the aseptic condition, repeating the step 2), inoculating the tumor blocks to the right inguinal part of a new mouse, and waiting for the next generation growth of PDX tumors;

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 60 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

When the tumor volume of the PDX inoculated mice reaches about 50mm ³, 40 animals with proper tumor volume are selected from 60 animals, and are randomly grouped according to the tumor volume, wherein n=8, so that all groups are ensured to 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. Tumor volume is expressed in mm ³ and tumor measurement formula is as described above.

Results

The experimental results are shown in FIG. 1. 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 Bonferroni method to see if there were significant differences between the different groups. * : p <0.05; * P <0.01. Figure 1 shows that tumor volume increase was significantly inhibited in the TIL-CTRL dosed mice compared to PBS-injected control tumor-bearing mice by day 40 post-dosing. Compared with the TIL-CTRL administration group, the tumor volumes of the tumor-bearing mice in the TIL-TBDR-7 and TIL-TBDR-15 administration groups are further significantly or very significantly reduced. And compared with TIL-TBDR-15, the TIL-TBDR-7 has more remarkable inhibition on the PDX tumor of the mice. 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 more obviously on the basis after the transgenic modification of TBDR-7 or TBDR-15, so that TBDR can obviously activate immune effector cells and improve the tumor killing capacity of the immune effector cells.

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 (28)

1. An isolated fusion protein consisting of the following from N-terminus to C-terminus: (1) an extracellular region consisting of an anti-TGF-beta antibody or an antigen-binding fragment thereof, and (2) a transmembrane region or a GPI anchor region, or (1) a membrane surface tag, and (2) an extracellular region consisting of an anti-TGF-beta antibody or an antigen-binding fragment thereof, and (3) a transmembrane region or a GPI anchor region, or (1) an extracellular region consisting of an anti-TGF-beta antibody or an antigen-binding fragment thereof, and (2) a membrane surface tag, and (3) a transmembrane region or a GPI anchor region, in, The membrane surface tag is the BCMA extracellular domain, and/or the membrane surface tag further comprises a linker located at the N-terminus or C-terminus of the BCMA extracellular domain, The fusion protein does not contain the intracellular domain or intracellular signal transduction region of the co-stimulatory signal molecule, The anti-TGF-beta antibody has the following HCDRs and LCDRs: the amino acid sequence of HCDR1 is shown in SEQ ID NO:48, the amino acid sequence of HCDR2 is shown in SEQ ID NO:49, the amino acid sequence of HCDR3 is shown in SEQ ID NO:50, the amino acid sequence of LCDR1 is shown in SEQ ID NO:51, the amino acid sequence of LCDR2 is shown in SEQ ID NO:52, the amino acid sequence of LCDR3 is shown in SEQ ID NO:53, The transmembrane region is the transmembrane region of CD28, The GPI anchor region is the GPI anchor domain of the CD52 protein, and The amino acid sequence of the CD28 transmembrane region is shown in SEQ ID NO:8, the amino acid sequence of the GPI anchor domain of the CD52 protein is shown in SEQ ID NO:12, the amino acid sequence of the BCMA extracellular domain is shown in SEQ ID NO:22, and the amino acid sequence of the linker is shown in SEQ ID NO:24 or 26. 2. An isolated fusion protein consisting of, from N-terminus to C-terminus, an extracellular region consisting of an anti-TGF-beta antibody or an antigen-binding fragment thereof, and an IL-7Ralpha transmembrane region, in, The anti-TGF-beta antibody has the following HCDRs and LCDRs: the amino acid sequence of HCDR1 is shown in SEQ ID NO:48, the amino acid sequence of HCDR2 is shown in SEQ ID NO:49, the amino acid sequence of HCDR3 is shown in SEQ ID NO:50, the amino acid sequence of LCDR1 is shown in SEQ ID NO:51, the amino acid sequence of LCDR2 is shown in SEQ ID NO:52, the amino acid sequence of LCDR3 is shown in SEQ ID NO:53, and The amino acid sequence of the IL-7Ralpha transmembrane region is shown in SEQ ID NO:10. 3. The fusion protein according to claim 1 or 2, wherein the anti-TGF-beta antibody comprises any one or more selected from single-chain antibodies, Fab, Fab' and F(ab')2. 4. The fusion protein according to claim 3, characterized in that the amino acid sequence of VH of the anti-TGF-beta antibody is as shown in SEQ ID NO:46, and/or the amino acid sequence of VL of the antibody is as shown in SEQ ID NO:47. 5 . The fusion protein according to claim 4 , wherein the anti-TGF-beta antibody is a single-chain antibody shown in SEQ ID NO: 4. The fusion protein according to claim 5 , wherein the fusion protein further comprises a signal peptide. 7. The fusion protein according to claim 6, wherein the signal peptide is a CD8 signal peptide, a TBRII signal peptide or a CD52 signal peptide. 8. The fusion protein according to claim 7, wherein the signal peptide has the amino acid sequence described in SEQ ID NO: 2, 6 or 28. 9. A polynucleotide molecule having a nucleic acid sequence encoding the fusion protein according to any one of claims 1 to 8. 10 . The polynucleotide molecule according to claim 9 , wherein the coding sequence of the anti-TGF-beta antibody in the fusion protein is as shown in SEQ ID NO: 3. 11. The polynucleotide molecule according to claim 9, characterized in that, in the fusion protein, the transmembrane region is the CD28 transmembrane region, and its coding sequence is shown in SEQ ID NO:7. 12. The polynucleotide molecule according to claim 9, characterized in that in the fusion protein, the transmembrane region is the IL7Ralpha transmembrane region, and its coding sequence is shown in SEQ ID NO:9. 13. The polynucleotide molecule according to claim 9, characterized in that, in the fusion protein, the GPI anchor region is the GPI anchor domain of CD52, and its coding sequence is shown in SEQ ID NO:11. 14. The polynucleotide molecule according to claim 9, wherein the fusion protein comprises a membrane surface tag, and the coding sequence of the BCMA extracellular domain of the membrane surface tag is shown in SEQ ID NO: 21. 15. The polynucleotide molecule according to claim 9, characterized in that the polynucleotide molecule is selected from any one of SEQ ID NOs: 31-45 and SEQ ID NOs: 54-55. A nucleic acid construct comprising the polynucleotide molecule according to claim 15 . 17. nucleic acid construct as claimed in claim 16, characterized in that the nucleic acid construct is a vector. 18. The nucleic acid construct of claim 17, wherein the nucleic acid construct is a non-viral vector. 19. The nucleic acid construct of claim 18, wherein the nucleic acid construct is an expression vector or an integration vector. 20. A genetically engineered cell, which expresses the fusion protein according to any one of claims 1 to 8, and/or carries the coding sequence of the fusion protein. 21. The cell according to claim 20, wherein the cell is an immune cell. 22. The cell of claim 21, wherein the immune cell comprises a T cell or a NK cell. 23. The cell of claim 21, wherein the immune cell is selected from the group consisting of: DN T cells, TCR-T, CAR-T, CAR-NK, CIK, NKT and TIL. 24. The cell of claim 22, wherein the cell further expresses CAR, or carries a coding sequence for CAR. 25. The cell of claim 22, wherein the cell further expresses an exogenous TCR, or carries a coding sequence of an exogenous TCR. 26. A method for sorting cells expressing the fusion protein of any one of claims 1 to 8, comprising detecting the anti-TGF-beta antibody or antigen-binding fragment thereof or the membrane surface label on the cell surface. 27. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and any one or more of the fusion protein of any one of claims 1-8, the polynucleotide molecule of any one of claims 9-15, the nucleic acid construct of any one of claims 16-19, and the cell of any one of claims 20-24. 28. Use of the fusion protein of any one of claims 1-8, the polynucleotide molecule of any one of claims 9-15, the nucleic acid construct of any one of claims 16-19, and the genetically engineered cell of any one of claims 20-24 in the preparation of a medicament for treating or preventing cancer, wherein the tumor cells of the cancer secrete TGF-beta, and the cancer is selected from: breast cancer, colon cancer, prostate cancer, melanoma, ovarian cancer, cervical cancer, kidney cancer, lung cancer, gastric cancer, liver cancer, pancreatic cancer, bile duct cancer and thyroid cancer.