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CN116640226B - Chimeric receptor for inhibiting host anti-exogenous immune cell HVG reaction - Google Patents

Chimeric receptor for inhibiting host anti-exogenous immune cell HVG reaction Download PDF

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CN116640226B
CN116640226B CN202211563854.1A CN202211563854A CN116640226B CN 116640226 B CN116640226 B CN 116640226B CN 202211563854 A CN202211563854 A CN 202211563854A CN 116640226 B CN116640226 B CN 116640226B
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CN116640226A (en
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刘靖
张世都
叶群瑞
董军纪
宋益哲
李凯峰
李利佳
李文佳
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Guangdong HEC Pharmaceutical
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Abstract

The present invention relates to a chimeric receptor for inhibiting host anti-foreign immune cell HVG response, comprising: a first extracellular domain comprising a domain that specifically targets 4-1 BB; a first transmembrane domain; the chimeric receptor does not include an intracellular domain; or further comprising a first intracellular domain comprising a first co-stimulatory domain and not comprising an activation signal transduction domain. When the chimeric receptor is co-expressed with a CAR molecule in immune cells such as T cells, the in vivo persistence is high, and the killing persistence of the CAR positive immune cells on 4-1-BB positive NK/T cells and CAR target antigen positive cells is enhanced. Furthermore, immune cells co-expressing chimeric receptor CR and CAR molecules show enhanced tumor killing ability, better CAR-T in vivo persistence and reduced HVG response.

Description

Chimeric receptor for inhibiting host anti-exogenous immune cell HVG reaction
Technical Field
The invention relates to the technical field of biology, in particular to a chimeric receptor for inhibiting host anti-exogenous immune cell HVG reaction.
Background
When allogeneic cells are not subjected to allogeneic transplantation through HLA-matched allogeneic cells, immune cells of a transplanted Host can generate immune rejection (Host anti-Graft, HVG, host versus Graft reaction) on the xenogeneic cells, and NK and T cells attack the allogeneic cells, so that the transplanted cells die and cannot exert cell functions for a long time. At present, allogeneic organ transplantation mainly depends on HLA matching to reduce immune rejection, and immunosuppressant is taken to inhibit acute rejection and chronic rejection.
In the use of generalized CAR-T, no way to solve HVG problems using immunosuppressants is possible. Existing protocols such as knockout of T cell HLA-related genes do not eliminate HVG responses: after B2M gene is knocked out, the CAR-T cells can avoid host T cell attack, but cannot avoid NK cell attack.
Under the condition that the HVG problem is not solved, the in vivo duration time of the existing general CAR-T cells in patients with FCA (fludarabine, cyclophosphamide and alemtuzumab) deep stranguria is only about 1 month, the in vivo duration time of the personalized CAR-T for one year or more can not be reached, the overall treatment effect of the general CAR-T cells is not ideal, and the recurrence rate of the patients after treatment is higher.
Disclosure of Invention
The invention aims to provide a chimeric receptor for inhibiting host anti-exogenous immune cell HVG reaction, which specifically recognizes a 4-1-BB target and does not comprise an activation signal transduction domain, so that the problem of strong intracellular signal transduction when the chimeric receptor is coexpressed with CAR is avoided, and the killing persistence of 4-1-BB positive NK/T cells can be effectively improved.
To this end, a first aspect of the invention provides a chimeric receptor (Chimeric Receptor, CR) comprising:
(i) A first extracellular domain comprising a domain that specifically targets 4-1 BB; and
(Ii) A first transmembrane domain;
the chimeric receptor does not include an intracellular domain; or alternatively
The chimeric receptor further comprises (iii) a first intracellular domain comprising a first co-stimulatory domain and no stimulatory signaling domain.
In some embodiments, the chimeric receptor comprises: a first extracellular domain comprising a domain that specifically targets 4-1 BB; a first transmembrane domain; the chimeric receptor does not include an intracellular domain.
In some embodiments, the chimeric receptor comprises: a first extracellular domain comprising a domain that specifically targets 4-1 BB; a first transmembrane domain; a first intracellular domain comprising a first co-stimulatory domain and not comprising a stimulatory signaling domain.
Further, the domain specifically targeting 4-1BB comprises an anti-4-1 BB antibody or antigen-binding fragment thereof, or a 4-1BBL extracellular domain or variant thereof.
Further, the anti-4-1 BB antibody or antigen-binding fragment thereof is selected from the group consisting of: fab 'fragments, F (ab') 2 fragments, bispecific Fab dimer (Fab) 2, trispecific Fab trimer (Fab) 3, fv, single chain Fv proteins (scFv), diabodies, triabodies, tetrabodies, disulfide stabilized Fv proteins (dsFv) and single domain antibodies (sdAb, camelid VHH, nanobody).
Further, the anti-4-1 BB antibody is a humanized antibody or an antigen-binding fragment thereof.
Further, the 4-1BBL is human 4-1BBL.
In some embodiments, the 4-1BBL extracellular domain comprises the amino acid sequence set forth in SEQ ID NO:1, and a polypeptide having the amino acid sequence shown in 1.
Further, the first extracellular domain further comprises a first hinge region, the first hinge region being linked to the first transmembrane domain.
In some embodiments, the first hinge region comprises one or a combination of two or more hinge regions selected from the group consisting of: igG, CD8, CD28.
In some embodiments, the first hinge region comprises a CD8 hinge region comprising the amino acid sequence set forth in SEQ ID NO:2, and a polypeptide having the amino acid sequence shown in 2.
In some embodiments, the first hinge region comprises a CH3 domain of IgG comprising a sequence as set forth in SEQ ID NO:3, and a polypeptide having the amino acid sequence shown in 3.
In some embodiments, the first hinge region comprises the CH3 domain of IgG and the CD8 hinge region.
In some embodiments, the first hinge region comprises the CH3 domain of IgG and the CD8 hinge region.
In some embodiments, the first transmembrane domain is selected from the group consisting of transmembrane domains of proteins of the following group: CD4, CD8, CD28, CD3 ζ, CD34.
In some embodiments, the first transmembrane domain comprises a CD8 transmembrane domain comprising SEQ ID NO:4, and a polypeptide having the amino acid sequence shown in (a) and (b).
Further, the first costimulatory domain is selected from one or more of the costimulatory domains of proteins of the group: CD28, ICOS, 4-1BB, OX20, OX40, CD27, CD30, HVEM.
In some embodiments, the first costimulatory domain comprises a 4-1BB costimulatory domain comprising the amino acid sequence as set forth in SEQ ID NO: 5.
Further, the first intracellular domain does not include an intracellular domain from cd3ζ.
Further, the chimeric receptor also includes a first signal peptide or a first leader sequence.
In some embodiments, the first signal peptide or first leader sequence comprises the sequence set forth in SEQ ID NO:6, and a polypeptide having the amino acid sequence shown in FIG. 6.
Still further, the chimeric receptor molecule further comprises a linker having an amino acid sequence as set forth in SEQ ID NO:21 or SEQ id no: 23.
In some embodiments, the chimeric receptor comprises: 4-1BBL extracellular domain or variant thereof, CH3 domain of IgG, CD8hinge region, CD8 transmembrane domain, 4-1BB co-stimulatory domain.
In some embodiments, the chimeric receptor comprises: 4-1BBL extracellular domain or variant thereof, CH3 domain of IgG, CD8 hinge region, CD8 transmembrane domain.
In some embodiments, the chimeric receptor comprises: 4-1BBL extracellular domain or variant thereof, CD8 hinge region, CD8 transmembrane domain.
In some embodiments, the chimeric receptor comprises: 4-1BBL extracellular domain or variant thereof, CD8 hinge region, CD8 transmembrane domain, 4-1BB co-stimulatory domain.
In some preferred embodiments, the chimeric receptor comprises:
(i) A first extracellular domain comprising a domain that specifically targets 4-1 BB; and
(Ii) A first transmembrane domain; and
(Iii) A first intracellular domain comprising a first costimulatory domain, and not comprising an activation signal transduction domain.
In a second aspect, the invention provides a combination of molecules comprising a chimeric receptor according to the first aspect of the invention, and a Chimeric Antigen Receptor (CAR); the chimeric antigen receptor comprises:
(iv) A second extracellular domain comprising an antigen binding domain;
(v) A second transmembrane domain; and
(Vi) A second intracellular domain comprising a second co-stimulatory domain, and a stimulatory signaling domain.
When allogeneic CART cells are transplanted, the immune system of the recipient is activated, so that the recipient has immune rejection reaction against xenogeneic grafts (CART), and the immune rejection reaction is mainly represented by that part of T cells of the recipient attack the allogeneic CART cells. The applicant finds that CR-expressing CAR-T cells can effectively attack and clear heterologous T cells activated by alloantigen in the research process, so that after the CAR-T cells are subjected to allogeneic transplantation, immune rejection mediated by T cells of a receiver can be effectively avoided, the in vivo persistence and cell functions of the allogeneic CAR-T are improved, and the tumor treatment effect is further enhanced.
Further, the antigen binding domain is an antibody or antigen binding fragment thereof.
Further, the antigen binding domain comprises an antibody or antigen binding fragment thereof selected from the group consisting of: fab 'fragments, F (ab') 2 fragments, bispecific Fab dimers (Fab 2), trispecific Fab trimers (Fab 3), fv, single chain Fv proteins (scFv), diabodies, triabodies, tetrabodies, disulfide stabilized Fv proteins (dsFv) and single domain antibodies (sdAb, camelid VHH, nanobodies).
Further, the antigen binding domain is a humanized antibody or antigen binding fragment thereof.
Further, the antigen binding domain binds an antigen selected from the group consisting of: tumor Associated Antigens (TAA), tumor Specific Antigens (TSA).
Further, the antigen binding domain binds an antigen selected from the group consisting of: b cell maturation antigen (BCMA)、CD4、CD7、CD16、CD19、CD20、CD22、CD30、CD33、CD37、CD38、CD44、CD44v6、CD44v7/8、CD70、CD79a、CD79b、CD123、CD133、CD138、CD171、 carcinoembryonic antigen (CEA).
In some embodiments, the antigen binding domain binds CD19, CD20, CD22, BCMA.
In some embodiments, the antigen binding domain comprises the amino acid sequence as set forth in SEQ ID NO: 7.
Further, the second extracellular domain further comprises a second hinge region, the second hinge region being linked to the second transmembrane domain.
In some embodiments, the second hinge region comprises one or a combination of two or more hinge regions selected from the group consisting of: igG, CD8, CD28.
In some embodiments, the second hinge region comprises a CD8 hinge region comprising the amino acid sequence set forth in SEQ ID NO:2, and a polypeptide having the amino acid sequence shown in 2.
In some embodiments, the second transmembrane domain is selected from the group consisting of transmembrane domains of proteins of the following group: CD4, CD8, CD28, CD3 ζ.
In some embodiments, the second transmembrane domain comprises a CD8 transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:4, and a polypeptide having the amino acid sequence shown in (a) and (b).
Further, the second co-stimulatory domain is selected from the group consisting of co-stimulatory domains of proteins of the group consisting of: CD28, ICOS, 4-1BB, OX20, OX40, CD27, CD30, HVEM.
In some embodiments, the second co-stimulatory domain comprises a CD28 co-stimulatory domain comprising the amino acid sequence as set forth in SEQ ID NO:8, and a polypeptide having the amino acid sequence shown in FIG. 8.
Further, the stimulation signal transduction domain comprises an immune receptor tyrosine activation motif (ITAM).
Further, the stimulatory signaling domain is isolated from a protein or polypeptide selected from the group consisting of: fcrγ, fcrβ, cd3γ, cd3δ, cd3ε, cd3ζ, CD22, CD79a, CD79b, and CD66d.
In some embodiments, the stimulation signal transduction domain is isolated from cd3ζ.
In some embodiments, the stimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 9.
Further, the chimeric antigen receptor also includes a second signal peptide or a second leader sequence.
In some embodiments, the second signal peptide or second leader sequence comprises the amino acid sequence: SEQ ID NO:10.
In some embodiments, the chimeric antigen receptor comprises: a CD8 hinge region, a CD8 transmembrane domain.
In some embodiments, the chimeric antigen receptor comprises an antigen binding domain, a CD8 hinge region, a CD8 transmembrane domain, and a second intracellular domain; preferably, the antigen binding domain binds an antigen selected from the group consisting of: tumor-associated antigens, tumor-specific antigens; preferably, the antigen binding domain binds to an antigen ：BCMA、CD4、CD7、CD16、CD19、CD20、CD22、CD30、CD33、CD37、CD38、CD44、CD44v6、CD44v7/8、CD70、CD79a、CD79b、CD123、CD133、CD138、CD171、 carcinoembryonic antigen selected from the group consisting of; preferably, the second intracellular domain comprises a second co-stimulatory domain, and an activation signal transduction domain; preferably, the second co-stimulatory domain is selected from one or more of the co-stimulatory domains of the group of proteins: CD28, ICOS, 4-1BB, OX20, OX40, CD27, CD30, HVEM; preferably, the activation signal transduction domain is isolated from a protein or polypeptide selected from the group consisting of: fcrγ, fcrβ, cd3γ, cd3δ, cd3ε, cd3ζ, CD22, CD79a, CD79b, and CD66d.
In some preferred embodiments, the chimeric antigen receptor comprises: binding to tumor antigen binding domain, CD8 hinge region, CD8 transmembrane domain, CD28 costimulatory domain, cd3ζ signaling domain.
In some more preferred embodiments, the chimeric antigen receptor comprises: an antigen binding domain that binds CD19, a CD8 hinge region, a CD8 transmembrane domain, a CD28 costimulatory domain, a cd3ζ signaling domain.
It should be noted that the CR molecules of the present invention can be co-expressed with CAR molecules of any structure in T cells, and are not limited to the CAR molecules described above, as long as they can achieve any of the technical effects of the present invention, such as enhanced tumor killing ability, better CAR-T in vivo persistence, reduced HVG response, or reduced tumor recurrence rate, which are all within the scope of the present invention.
In a third aspect of the invention there is provided a nucleic acid molecule encoding a chimeric receptor according to the first aspect of the invention and/or a combination of molecules according to the second aspect of the invention.
In a fourth aspect of the invention there is provided a vector comprising a chimeric receptor according to the first aspect of the invention and/or a combination of molecules according to the second aspect of the invention.
Further, the vector is a viral vector or a non-viral vector.
Further, the viral vector is selected from the group consisting of: adenovirus vectors, retrovirus vectors, vaccinia virus vectors, poxvirus vectors, adeno-associated virus vectors, herpes simplex virus vectors, lentivirus vectors, baculovirus vectors, sendai virus vectors, measles virus vectors, simian virus vectors.
In a fifth aspect of the invention there is provided a cell comprising a chimeric receptor according to the first aspect of the invention, a combination of molecules according to the second aspect of the invention, a nucleic acid molecule according to the third aspect of the invention, or a vector according to the fourth aspect of the invention.
Further, the cell is a eukaryotic cell or a prokaryotic cell.
Further, the cell is an immune cell.
Further, the cell is selected from the group consisting of: t cells, B cells, NK cells, neutrophils, monocytes, macrophages.
In some embodiments, the immune cell is a T cell.
In some embodiments, the immune cell is a Chimeric Antigen Receptor (CAR) transduced T cell (CAR-T).
In some embodiments, the immune cell is a T cell transduced by a T cell receptor (TCR-T). Further, the immune cells lack endogenous expression of TRAC, TRBC, 4-1BB and/or CD3 proteins; preferably, the cells lack endogenous expression of TRAC and/or 4-1BB proteins. Applicants have found during the course of the study that CR-CAR T cells have some degree of self-killing capacity, resulting in a decrease in their proliferation and expansion capacity, and that CR-CAR T cells may have a return in proliferation capacity after further knockout of the 4-1BB gene.
According to the technical scheme of the invention, when the chimeric receptor and the chimeric antigen receptor are expressed in T cells with TRAC, TRBC or CD3 gene knocked out, the CAR-T cells which are universal in the real sense can be obtained. This type of cell does not express TCR molecules, is GVHD-free (graft versus host response), and is able to avoid HVG responses.
It is specifically contemplated that the chimeric molecules (CR) of the invention may in some cases be extended to be expressed in T cell receptor chimeric T cells (TCR-T).
In a sixth aspect of the invention there is provided a pharmaceutical composition comprising a cell according to the fifth aspect of the invention and a pharmaceutically acceptable carrier.
In a seventh aspect, the invention provides the use of a chimeric receptor according to the first aspect, a combination of molecules according to the second aspect, a nucleic acid molecule according to the third aspect, a vector according to the fourth aspect, a cell according to the fifth aspect or a pharmaceutical composition according to the sixth aspect for the preparation of a medicament for inhibiting immune rejection.
According to an eighth aspect of the present invention there is provided the use of a chimeric receptor according to the first aspect, a combination of molecules according to the second aspect, a nucleic acid molecule according to the third aspect, a vector according to the fourth aspect, a cell according to the fifth aspect or a pharmaceutical composition according to the sixth aspect for the preparation of a medicament for the treatment of cancer.
Further, the cancer includes liver cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, brain cancer, head and neck cancer, bone cancer, thyroid cancer, kidney cancer, skin cancer, leukemia, lymphoma, multiple myeloma, or the like.
Compared with the prior art, the technical scheme of the invention has the following steps:
The invention provides a novel chimeric molecule-chimeric receptor CR molecule which specifically recognizes a 4-1BB target, does not comprise an activation signal transduction domain, has stronger in vivo persistence when being coexpressed with a CAR molecule in immune cells such as T cells, and enhances the killing persistence of the CAR positive immune cells on 4-1-BB positive NK/T cells and CAR target antigen positive cells.
The invention also provides a molecular combination comprising the chimeric receptor and the chimeric antigen receptor, and corresponding vectors, cells, etc. When the CR molecule and CAR are co-expressed by cells (e.g., T cells), they are able to specifically recognize and kill 4-1-BB positive T/NK cells and have good persistence, thus solving HVG problems arising from allograft transplantation. On the other hand, cells co-expressing CR molecules and CAR molecules exhibit enhanced tumor killing capabilities, including being able to recognize tumor cells of low antigen abundance, better CAR-T metabolism and differentiation phenotype, better CAR-T in vivo persistence, thereby increasing general CAR-T cell in vivo persistence time, reducing tumor recurrence rate.
Drawings
Fig. 1: CR#1 molecular structure diagram;
fig. 2: CR#2 molecular structure diagram;
fig. 3: CR#3 molecular structure diagram;
fig. 4: CR#4 molecular structure diagram;
fig. 5: CD19-CAR molecular structure;
Fig. 6: expression of cr#1, cr#4 molecules and FMC63-CAR in T primary cells;
fig. 7: CD137 expression after 48 hours of stimulation of CD19-UCART cells;
Fig. 8: residual target cell ratio after 48 hours killing of different CR (cr#1-cr#4) CAR-T to 4-1BB + cells;
Fig. 9: UCART cells expressing each CR molecule (CD 3 - cells) remained after 7 days incubation with heterologous T cells;
fig. 10: 4-1BB positive cell ratio after T cells were electrotransferred at different doses of sgRNA-CAS 9;
Fig. 11: comparison of proliferation of CAR-T expressing CR#1 molecule and CR#1-CAR-T after 4-1BB knocked out in the course of magnetic bead stimulation, CAR-T being control group;
Fig. 12: the proliferation of CAR-T expressing CR#1 molecule and CR#1-CAR-T after knocking out 4-1BB was compared during the subsequent amplification of the magnetic bead stimulation stage, and CAR-T was used as control group.
Detailed Description
Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.
Definition of the definition
Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly used in the art to which this invention belongs. For the purposes of explaining the present specification, the following definitions will apply, and terms used in the singular will also include the plural and vice versa, as appropriate.
As used herein, the terms "4-1BB" and "4-1BB receptor" have the same meaning and are used interchangeably, and refer to any form of the 4-1BB receptor, as well as variants, isoforms, and species homologs that retain at least a portion of the activity of the 4-1BB receptor, also known as CD137 or TNFRSF 9. In some embodiments, the 4-1BB comprises the native sequence 4-1BB from all mammalian species, such as human, canine, feline, equine, and bovine; in other embodiments, the 4-1BB is from a human.
As used herein, the term "anti-4-1 BB antibody" refers to an antibody that is capable of specifically recognizing and binding to 4-1 BB.
As used herein, the term "4-1BBL", also known as 4-1BB ligand, CD137L or TNFSF9, is a high affinity ligand for the 4-1BB receptor, with the receptor binding site on 4-1BBL being located in its extracellular domain. Taking human 4-1BBL (NP-003802.1) as an example, in some embodiments of the invention, a first extracellular domain comprising the extracellular domain of human 4-1BBL is provided, and a polypeptide as set forth in SEQ ID NO:1, and a polypeptide having the amino acid sequence shown in 1.
The present disclosure provides modified immune cells comprising a Chimeric Antigen Receptor (CAR), or T Cell Receptor (TCR), which exhibit specific binding to a neoantigen. The CAR may comprise an antigen interaction domain capable of binding to a T cell surface protein, a transmembrane domain and an intracellular signaling domain.
The antigen binding domain may comprise any protein or molecule capable of binding an antigen, such as a T cell surface protein. Non-limiting examples of antigen binding domains include, but are not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, murine antibodies, or functional derivatives, variants, or fragments thereof. Including, but not limited to, fab ', F (ab') 2, fv, single chain Fv (scFv), minibodies, diabodies, and single domain antibodies such as heavy chain variable domains (VH), light chain variable domains (VL) and variable domains (VHH) of camelid-derived nanobodies. In some embodiments, the first antigen binding domain comprises at least one of Fab, fab ', F (ab') 2, fv, and scFv. In some embodiments, the antigen binding domain comprises an antibody mimetic. An antibody mimetic refers to a molecule capable of binding a target molecule with an affinity comparable to that of an antibody, including single chain binding molecules, cytochrome b 562-based binding molecules, fibronectin or fibronectin-like protein scaffolds (e.g., adnectin), lipocalin scaffolds, calixarene scaffolds, a domain, and other scaffolds. In some embodiments, the antigen binding domain comprises a transmembrane receptor or any derivative, variant or fragment thereof. For example, an antigen binding domain.
In some embodiments, the antigen binding domain may comprise a scFV. The scFv may be derived from antibodies of known variable region sequences. In some embodiments, the scFv may be derived from antibody sequences obtained from available mouse hybridomas. scFv can be obtained from whole exon sequencing of tumor cells or primary cells. In some embodiments, the scFv may be altered. For example, the scFv may be modified in various ways. In some cases, the scFv may be mutated such that the scFv may have a higher affinity for its target. In some cases, the affinity of scFv for its target may be optimized for targets expressed at low levels on normal tissues. This optimization can be done to minimize potential toxicity, such as hypercytokinemia. In other cases, cloning of scFv with higher affinity for the target membrane-bound form may be preferred over its soluble form counterpart. This modification can be done if certain targets can also be detected in soluble forms at different levels, and their targeting can cause unintended toxicity, such as hypercytokinemia.
The antigen binding domain of the CAR of the present system can be linked to an intracellular signaling domain by a transmembrane domain. The transmembrane domain may be a transmembrane segment. The transmembrane domain of the subject CAR may anchor the CAR to the plasma membrane of a cell, e.g., an immune cell. In some embodiments, the transmembrane segment comprises a polypeptide. The transmembrane polypeptide that connects the antigen binding domain and the intracellular signaling domain of the CAR can have any suitable polypeptide sequence. In some cases, the transmembrane polypeptide comprises a polypeptide sequence of the transmembrane portion of an endogenous or wild-type transmembrane protein. In some embodiments, a transmembrane polypeptide comprises a polypeptide sequence having at least 1 (e.g., at least 2,3,4,5,6,7,8,9, 10 or more) amino acid substitutions, deletions, and polypeptides. Insertion is compared to the transmembrane portion of the endogenous or wild-type transmembrane protein. In some embodiments, the transmembrane polypeptide comprises a non-native polypeptide sequence, such as a sequence of a polypeptide linker. The polypeptide linker may be flexible or rigid. The polypeptide linker may be structured or unstructured. In some embodiments, the transmembrane polypeptide transmits a signal from an extracellular region of the cell to an intracellular region through the antigen binding domain. The natural transmembrane portion of CD28 is available for CAR. In other cases, the natural transmembrane portion of CD8 a can also be used for the CAR.
In one aspect, the present disclosure provides a Universal CAR (UCAR) immune cell characterized in that a. The UCAR immune cell surface expresses a Chimeric Antigen Receptor (CAR); b. the UCAR immune cells are used for treating T cell tumor patients; c. the CAR of the UCAR immune cell comprises an antibody or variable region of an antibody that targets a T cell surface protein; d. the UCAR immune cells are not from the patient's body; in some embodiments, the UCAR immune cells are UCAR-T cells.
In some embodiments, UCAR-T cells knock out all or part of the TCR gene (e.g., TRAC), or all or part of the HLA gene (e.g., B 2 M). The gene is knocked out, so that UCAR-T cells can be prevented from attacking autologous cells of a patient on one hand, and UCAR-T cells can be prevented from being attacked by autologous T cells of the patient on the other hand.
In some embodiments, UCAR-T cells of the invention knock out the TRAC gene; in other embodiments, UCAR-T cells knock out both TRAC and 4-1BB genes.
In some embodiments, the gene knockout employs a zinc finger ribonuclease (ZFN) technology system; in some embodiments, gene knockout employs a transcription activator-like effector nuclease (TALEN) technology system; in some embodiments, gene knockout employs a CRISPR system; in some embodiments, gene knockout employs a CRISPR-Cas9 system.
As used herein, the term "amino acid" refers to a naturally occurring group of carboxyα -amino acids comprising alanine (three letter code: ala, one letter code: a), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (gin, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y) and valine (Val, V).
As used herein, the term "vector" refers to a nucleic acid molecule for introducing a particular gene operably associated therewith into a target cell and directing expression of the particular gene. The term includes vectors that are self-replicating nucleic acid structures and that are incorporated into the genome of a host cell into which they have been introduced. The expression vector of the present invention includes an expression cassette. Expression vectors allow for the transcription of a large number of stable mRNAs. Once the expression vector enters the target cell, ribonucleic acid molecules or proteins encoded by the gene are produced by cellular transcription and/or translation mechanisms. Vectors useful in the present invention include, but are not limited to: viral vectors, such as adenovirus, retrovirus, vaccinia virus, poxvirus, adeno-associated virus, herpes simplex virus, lentivirus, baculovirus, sendai virus, measles virus, simian virus vectors; non-viral vectors such as plasmids, lipid complexes (cationic liposome-DNA complexes), multimeric complexes (cationic polymer-DNA complexes), protein-DNA complexes, and the like.
As used herein, the term "host cell" refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells" which include the primary transformed cell and the progeny derived therefrom, regardless of the number of passages. The offspring may not be exactly identical in nucleic acid content to the parent cell, but may contain mutations. Mutant offspring that have the same function or biological activity as screened or selected in the original transformed cell are included herein. In embodiments of the invention, the host cell may be an immune cell.
As used herein, the term "immune cell" refers to a cell of the immune system that can be classified as a lymphocyte (e.g., T cell, B cell, and NK cell), neutrophil, monocyte, macrophage. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is an NK cell. In some embodiments, the immune cell is a macrophage. In some embodiments, the immune cell is an engineered immune cell, meaning that the immune cell has been genetically modified to express a non-naturally occurring protein (e.g., chimeric receptor, chimeric antigen receptor) or to comprise a foreign nucleic acid; and/or the immune cells have been modified to delete expression of proteins encoded by certain endogenous genes (e.g., TRAC gene, TRBC gene, CD3 gene).
The term "T cell" or "T lymphocyte" is art-recognized and is intended to include thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes or activated T lymphocytes. The T cells may be T helper (Th) cells, such as T helper 1 (Th 1) or T helper 2 (Th 2) cells. The T cells may be helper T cells (HTL; CD4 + T cells) CD4 + T cells, cytotoxic T cells (CTL; CD8 + T cells), tumor infiltrating cytotoxic T cells (TIL; CD8 + T cells), CD4 +CD8+ T cells, CD4 -CD8- T cells or any other T cell subpopulation.
As used herein, "CAR-T", "CART" refer to chimeric antigen receptor T cells.
As used herein, "CR-CART cells" and "CR-CAR-T cells" refer to CART or CAR-T cells that express CR molecules.
As used herein, "CR-UCART cells" and "CR-UCAR-T cells" refer to UCART or UCAR-T cells that express a CR molecule.
As used herein, the term "T cell receptor" (TCR) refers to a heterogeneous cell surface receptor capable of specifically interacting with a target antigen. As used herein, "TCR" includes, but is not limited to, naturally occurring and non-naturally occurring TCRs; full length TCR and antigen-binding portion thereof; chimeric TCRs; TCR fusion constructs; synthesizing TCR.
Example 1 construction and expression of CR receptor molecules and CAR molecules
1. The amino acid sequences of the different chimeric receptor proteins were designed as follows:
CR #1 receptor: the signal peptide +4-1-BBL protein (transmembrane domain removed) +CH3 finger+CD8finger+CD8TM (transmembrane domain) +4-1-BB costimulatory domain. The schematic structure of CR molecule is shown in figure 1.
CR #2 receptor: signal peptide +4-1-BBL protein (transmembrane domain removed) +CH3 finger+CD8finger+CD8TM (transmembrane domain). The molecular structure of the receptor is schematically shown in figure 2.
CR #3 receptor: the signal peptide +4-1-BBL protein (transmembrane domain removed) +CD8 finger+CD8TM (transmembrane domain) +4-1-BB costimulatory domain. The schematic structure is shown in fig. 3.
CR #4 receptor: signal peptide +4-1-BBL protein (removal of transmembrane domain) +CD8 finger+CD8TM (transmembrane domain). The schematic structure is shown in fig. 4.
CD19-CAR molecule: fmc63+cd8finger+cd8tm (transmembrane domain) +cd28 co-stimulatory domain or 4-1BB co-stimulatory domain+cd3zeta. See fig. 5.
The amino acid sequence and corresponding DNA sequence of the CR receptor molecule are shown in table 1:
table 1cr#1 receptor molecules:
Table 2 cr#2 molecules:
Table 3 cr#3 molecules:
Table 4 cr#4 molecules:
the amino acid sequence of the anti-CD19FMC-CAR with the signal peptide is as follows:
MGVKVLFALICIAVAEADIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSEFTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO：16)
the anti-CD19FMC-CAR DNA expression sequence with signal peptide is as follows:
ATGGGCGTCAAGGTCCTGTTCGCCCTGATCTGCATCGCCGTCGCCGAGGCCGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGATCACAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCAGAATTCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCTTCATCATCTTTTGGGTCCGCAGCAAGCGGAGCAGAGGCGGCCACAGCGACTACATGAACATGACCCCTAGACGGCCTGGCCCCACCAGAAAGCACTACCAGCCCTACGCCCCTCCCCGGGACTTTGCCGCCTACAGAAGCCGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGCCTGTATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCAAGG(SEQ ID NO：17) The designed nucleotide sequences of different CR structures and CD19-CAR are sent to BamH I and SalI double enzyme digestion after being synthesized by Suzhou gold intellectual biotechnology limited company, and then are connected into Piggybac expression vector pPBEF (self-constructed vector by the company), and are transferred into stbl3 competence to amplify and extract plasmids for standby.
2. T cell activation, electrotransformation of CR-PB plasmid
2.1 Taking 2mL of CD3 + positive cells (activity 98.5% and living cells 5X10 6), adding into a 15mL centrifuge tube containing 5mL AIM-V, centrifuging 500g and 5min to collect cell supernatant, re-suspending cells with 10mL AIM-V culture medium (containing 20IU/mL of IL-2), transferring into a 10cm plate for culturing, taking 180mL of CD3/28 magnetic beads, adding into 4mL buffer for washing, placing into a magnetic rack for 2min, removing supernatant, taking 180 mu L of AIM-V culture medium (containing 20IU/mL of IL-2) for re-suspension, adding into cell liquid, mixing, placing into an incubator for culturing, and carrying out virus transfection after culturing for 24 hours.
2.2 Cells were removed and placed in a 15mL centrifuge tube, placed in a magnetic rack for 2min, and the cell sap was aspirated into another clean 15mL centrifuge tube. 10. Mu.L of cell sap was taken, 10. Mu.L of AO/PI dye solution (staining solution) was added, mixed well, added to a cell counting plate, viability was 98.41%, viable cell density was 4.01X10 6, 3mL of cell sap was taken, centrifuged at 500g for 5min, and the supernatant was discarded.
After counting, determining the number of holes to be electrically turned, subpackaging the holes into 12-hole plates, and preheating 1.5mL of culture medium in each hole in an incubator at 37 ℃ for half an hour in advance;
2.3 preparation of electrotransfer solution (100. Mu.L System): 82 mu L P3PRIMARY CELL solution+18 mu L support, and mixed well for use. The electrotransformation kit is Lonza P3PRIMARY CELL electroporation kit.
Taking 5.0E+6 cells, subpackaging in 1.5mL EP tube, centrifuging at room temperature for 10min, discarding supernatant, washing with 1mL PBS (calcium and magnesium ion free), centrifuging at room temperature for 10min, discarding supernatant, and collecting supernatant;
Electric conversion: CR-PB plasmid (4. Mu.g), CD19-CAR-PB plasmid (4. Mu.g) and Pbase plasmid (4. Mu.g) were added to 100. Mu.L of electrotransfer solution, and after gentle mixing, the cells were resuspended and transferred into electrotransfer cups for electrotransfer procedure FI-115. After electric shock, the cells are transferred into a 12-hole plate which is incubated with 1.5mL of culture medium in advance, then 500 mu L of culture medium is taken for washing an electrorotating cup, and the cell fluid is transferred into the 12-hole plate and placed into a carbon dioxide incubator for culture.
Cells were centrifuged to remove the electrotransfer solution 12 hours after electrotransfer, and plated at 10-6 cells/mL density until day three (day of enrichment) for CAR and CR plasmid expression assays.
3. Flow detection of CAR and CR molecule expression
3.1 Different sets of CAR-T cells were collected, about 10 6 per set, totaling 1 negative control T cell and 10 experimental sets.
3.2 Centrifuging 500g for 5min to collect cells, and discarding the supernatant; add 500. Mu.L PBS to resuspend, centrifuge 500g,5min, repeat 2 times, discard supernatant. The negative control group was resuspended in 200. Mu.L PBS and stored at 4 ℃.
3.3 Experimental groups were resuspended by adding 500. Mu. LPBS, 1. Mu.L of FTIC-labeled anti-FMC63 antibody were added and incubated at 4℃for 30min in the absence of light; after incubation, 4 ℃,500g,5min, and centrifuging to collect cell waste supernatant; add 500. Mu.L PBS to resuspend, 4 ℃,500g,5min, collect cell supernatant by centrifugation, repeat 3 times, add 200. Mu.L PBS to resuspend for flow-through detection of CAR expression.
3.4 Experiments were resuspended in 200. Mu.L PBS, added with 0.4. Mu. L R-Phycoerythrin affinipure goat anti-human IgG Fcgamma FRAGMENT SPECIFIC antibody and incubated for 20min in the dark; after the incubation is completed, 500g and 5min are carried out, and cell supernatant is collected by centrifugation; add 500. Mu.L PBS to resuspend, 4 ℃,500g,5min, collect cell supernatant by centrifugation, repeat 3 times, add 200mL PBS to resuspend, for flow assay CR molecule expression.
By means of Piggy-bac plasmid combined electrotransformation, the CAR molecule and CR molecule transduction can be realized, and each protein can be expressed in human T cells and correctly positioned on cell membranes. The results are shown in FIG. 6.
Flow results showed that 5 molecules could be localized at the cell membrane and detected using live cell staining flow.
Example 2 CR-detection of the killing ability of CAR T cells on 4-1BB + cells
1. Antigen activation of CD19-UCAR-T cells to express 4-1BB
1.1 Preparation of CD19 antigen coated well plates: 2. Mu.g/mL CD19 protein 2 mL/well was coated overnight or room temperature in 6 well plates at 4℃for 6 hours. After the coating, sucking away the protein, and washing once with PBS for standby;
1.2 resuscitating CD19-UCART cells, and culturing the 2.0E+6 total cells in a 2mLCTS TMOpTmizerTM T Cell Expansion medium for 48 hours after adding the cells into the coated wells;
1.3 1.0E+6 cells were removed and 4-1BB expression rate flow assay was performed: 350g, centrifuging and collecting cells in 5min, and discarding the supernatant; add 500. Mu.L PBS to resuspend, centrifuge 350g,5min, repeat 2 times, discard supernatant. After 200. Mu. L STAINING buffer was resuspended, 20. Mu.L of anti-human CD137 antibody was added and incubated at room temperature for 20min in the absence of light. Add 500 μl PBS to resuspend, centrifuge 350g,5min, repeat 2 times, finally resuspend incubated cells on-machine with 200 μl PBS. The flow chart is shown in FIG. 7, the positive rate is 67.40%, the unstimulated UCAR-T is the control group, the positive rate is 5.76%, and the CD19 stimulation is proved to be effective.
2. CR-CAR-T and 4-1BB + UCAR-T are incubated together, and the killing effect is detected.
2.1 Co-incubating UCAR-T after stimulation with CR-expressing CAR-T cells in an amount of 1:1 for CR +/CD137+, and incubating the non-CR-expressing CAR-T with UCAR-T after stimulation in a control group. A separate incubation group was also established as a control. Co-incubation wells with 6.0E+4/well added per well CR-CAR-T, CD137 + UCAR-T also 6.0E+4/well; the separate incubation groups were then stimulated with only UCAR-T or CD19 antigen, respectively.
2.2 After 48 hours incubation, total cell counts were performed and staining was performed with CD3/CAR antibody. CD3 -CAR+ cells are target cells UCAR-T and CD3 + cells are CR-CAR-T cells. Comparison of the number of CD3 -CAR+ cells in each co-incubated group after 48 hours with the number of CD3 -CAR+ cells in UCAR-T alone incubated group resulted in a ratio of target UCART cell changes after co-incubation. The results are shown in FIG. 8.
The results show that all four CR-CART molecules have specific killing ability to 4-1BB + cells, and the CR#1-CART and CR#3-CART have the strongest killing ability.
Example 3 TCR ko ability of CR-CAR T cells to inhibit immune rejection of allogeneic T cells
1. TCR KO type CR-CAR T preparation
1.1 Resuscitating T cells, and 2.0E+6 total cells were resuspended in 2ml CTS TMOpTmizerTM T Cell Expansion medium and added to the coated wells for 25 hours;
1.2 transduction of CR and CAR expression plasmids and TRAC-RNP Using an electrotransport apparatus to T cells
Preparing electrotransfer liquid (100 mu L system): 82 mu L P3 PRIMARY CELL solution+18 mu L support, and mixed well for use. The electrotransport kit was lonza P3. 3 PRIMARY CELL electroporation kit.
Taking 5.0E+6 cells, subpackaging in 1.5mL EP tube, centrifuging at room temperature for 10min, discarding supernatant, washing with 1mL PBS (calcium and magnesium ion free), centrifuging at 100g and RT for 10min, discarding supernatant;
Electric conversion: to 100. Mu.L of the electrotransfer solution were added CR-PB plasmid (4. Mu.g), CD19-CAR-PB plasmid (4. Mu.g) and Pbase plasmid (4. Mu.g), and 5. Mu.L of the electrotransfer solution was incubated with 4. Mu. G TRAC SGRNA +4. Mu.g CAS9 to incubate the RNP complex. (the sgRNA sequence is mA*mG*mA*GUCUCUCAGCUGGUACAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU*mU*mU*mU(SEQ ID NO：18)). after being gently mixed, the cells are resuspended and transferred into an electrorotating cup, the electrorotating program FI-115. After electric shock, the cells are transferred into a 12-well plate which is incubated with 1.5mL of culture medium in advance, then 500 mu L of culture medium is taken for washing the electrorotating cup, and the cell sap is transferred into the 12-well plate and placed into a carbon dioxide incubator for culture.
1.3 Removing electrotransfer solution 12 hours after electrotransfer, adding CD3/28 antibody magnetic beads (20 mu L magnetic beads 1E6 cells) to stimulate, which is marked as Day0, until Day2 removes the magnetic beads by using a magnet, and continuing culturing until D8. CR, CAR expression and CD3 - ratio were tested.
2. CR-UCAR-T is incubated with allogeneic T cells, and UCAR-T persistence is detected.
2.1 Recovery of heterologous T cells, culture was performed without activation following resuspension of 2.0E+6 total cells with 2mL CTS TMOpTmizerTM T Cell Expansion medium.
2.2 Allogeneic T cells were co-incubated with allogeneic T cells 24 hours after resuscitation at a ratio of CR-UCART cells to allogeneic T cells of 1:4, and the control group was a UCART co-incubated group with allogeneic T cells that did not express CR molecules. A separate incubation group was also established as a control. Co-incubation wells with 1.0E+5/well added per well CR-UCART, and 4.0E+5/well for heterologous T cells; only CR-CART or UCART was added to the separate incubation groups, and the control group UCART, which did not express CR molecules, was incubated with 4.0E+5 heterologous T cells, with the same total cell count as CR-UCART.
2.3 After 7 days of incubation, total cell counts were performed and staining with CD3/CR antibody was performed. CD3 + cells are allogeneic T cells and CD3 - cells are UCART cells. Comparison of UCART cell numbers in each co-incubated group can give a determination as to whether the expressed CR molecules are resistant to attack by allogeneic T cells on UCART. The results are shown in FIG. 9:
The results showed that UCAR T cells expressing CR molecules were resistant to the challenge of UCART cells by allogeneic T cells (CR#1 and #3 were more resistant and CR#2 and #4 were relatively weak), and that UCAR T (CD 3 - cells) from the control group, which did not express CR molecules, was almost absent.
Example 44-1 Effect of BB knockout on CR#1-CAR-T cell cognate killing
1. Preparation of 4-1 BBKO type CR#1-CAR-T
1.1 Design of the 4-1BB SgRNA sequences to be mA*mC*mA*UUUAACGAUCAGAAACGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU*mU*mU*Mu(SEQ ID NO：19)
1.2 Resuscitating T cells, and electrotransformation was performed after 2.0E+6 total cells in each well of 6 well plates were resuspended in 2ml CTS TMOpTmizerTM T Cell Expansion medium and cultured for 24 hours;
1.3 transduction of CR#1 and CAR expression plasmids to T cells Using an electrotransport apparatus, amplification
Preparing electrotransfer liquid (100 mu L system): 82 mu L P3 PRIMARY CELL solution+18 mu L support, and mixed well for use. The electrotransformation kit is Lonza P3 PRIMARY CELL electroporation kit.
Taking 5.0E+6 cells, subpackaging in 1.5ml EP tube, centrifuging at room temperature for 10min, discarding supernatant, washing with 1ml PBS (calcium and magnesium ion free), centrifuging at 100g and RT for 10min, discarding supernatant;
4-1BB KO CR-CART experimental group electrotransformation: mu.L of the electrotransfer solution was added with CR#1-PB plasmid (4. Mu.g), CD19-CAR-PB plasmid (4. Mu.g) and Pbase plasmid (4. Mu.g) and 5. Mu.L of the electrotransfer solution was incubated with 8. Mu. G TRAC SGRNA +8. Mu.g CAS9 to incubate the RNP complex. After gentle mixing, 5.0E+6 cells were resuspended and transferred to an electrorotor, electrotransfer procedure FI-115.
CAR-T control group electrotransformation: CD19-CAR-PB plasmid (4. Mu.g) and Pbase plasmid (4. Mu.g) were added to 100. Mu.L of the electrotransfer solution. After gentle mixing, 5.0E+6 cells were resuspended and transferred to an electrorotor, electrotransfer procedure FI-115.
CR-CART control group electric switching: to 100. Mu.L of electrotransfer solution were added CR#1-PB plasmid (4. Mu.g), CD19-CAR-PB plasmid (4. Mu.g) and Pbase plasmid (4. Mu.g). After gentle mixing, 5.0E+6 cells were resuspended and transferred to an electrorotor, electrotransfer procedure FI-115.
The effect of knockdown of 4-1BB on T cells after electrical transformation of sgRNA at different doses was searched for, see FIG. 10. As can be seen from the results of FIG. 10, the knockout efficiency of 4-1BB increased with increasing dose of sgRNA.
2. Proliferation assay of 4-1BB KO CR-CART cells, CR-CART control cells and CAR-T during D3-D12.
2.1 Removing electrotransfer solution 12 hours after electrotransfer, culturing for 24 hours, and adding CD3/28 antibody magnetic beads (25 mu L magnetic beads 1E6 cells) for stimulation, which is designated Day 3. Stimulation to Day 7 removed the beads.
2.2 Cell counts were taken daily and cell numbers at each point were recorded.
Transferring the cell suspension into a 15ml centrifuge tube, and centrifuging in 300g and 5min of the centrifuge; the supernatant was aspirated, the pellet of light cells was pelleted, 1ml of human fibroblast serum free complete medium was added to the centrifuge tube, and 20. Mu.l of cell suspension was resuspended in 1.5ml EP tube and counted by AO/PI 20. Mu.l (1:1) staining.
The results are shown in fig. 11 and 12. From the results, the proliferation potency of CR#1-CART was significantly weaker than that of CART group due to the cognate killing effect of CR molecules targeting 4-1BB + cells, but the proliferation potency of CR#1 type 4-1BB + KO also showed a significant return in case of lower knockout efficiency after 4-1BB knockout.
The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims (11)

1. A cell comprising a combination of molecules, a nucleic acid molecule encoding the combination of molecules, or a vector comprising the combination of molecules; the combination of molecules includes a chimeric receptor, and a chimeric antigen receptor;
The chimeric receptor is selected from the group consisting of:
The composition of the amino acid sequence is as follows: SEQ ID NO:6, the signal peptide shown in SEQ ID NO:1, a 4-1-BBL protein region shown in SEQ ID NO:21, a linker as set forth in SEQ ID NO:3, a CH3 domain shown in SEQ ID NO:23, a linker as set forth in SEQ ID NO:2, the CD8 hinge region shown in SEQ ID NO:4, a CD8 transmembrane domain shown in SEQ ID NO:5, a 4-1-BB costimulatory domain shown in figure 5;
The composition of the amino acid sequence is as follows: SEQ ID NO:6, the signal peptide shown in SEQ ID NO:1, a 4-1-BBL protein region shown in SEQ ID NO:21, a linker as set forth in SEQ ID NO:3, a CH3 domain shown in SEQ ID NO:23, a linker as set forth in SEQ ID NO:2, the CD8 hinge region shown in SEQ ID NO:4, a CD8 transmembrane domain shown in figure 4;
The composition of the amino acid sequence is as follows: SEQ ID NO:6, the signal peptide shown in SEQ ID NO:1, a 4-1-BBL protein region shown in SEQ ID NO:21, a linker as set forth in SEQ ID NO:2, the CD8 hinge region shown in SEQ ID NO:4, a CD8 transmembrane domain shown in SEQ ID NO:5, a 4-1-BB costimulatory domain shown in figure 5; or alternatively, the first and second heat exchangers may be,
The composition of the amino acid sequence is as follows: SEQ ID NO:6, the signal peptide shown in SEQ ID NO:1, a 4-1-BBL protein region shown in SEQ ID NO:21, a linker as set forth in SEQ ID NO:2, the CD8 hinge region shown in SEQ ID NO:4, a CD8 transmembrane domain shown in figure 4;
the chimeric antigen receptor comprises:
(iv) A second extracellular domain comprising an antigen binding domain;
(v) A second transmembrane domain; and
(Vi) A second intracellular domain comprising a second co-stimulatory domain, and an activation signal transduction domain;
The amino acid sequence of the chimeric antigen receptor comprises SEQ ID NO:16;
the cells are immune cells and lack endogenous expression of TCR, 4-1BB and/or CD3 proteins.
2. The cell of claim 1, wherein the vector is a viral vector or a non-viral vector.
3. The cell of claim 2, wherein the viral vector is selected from the group consisting of: adenovirus vectors, retrovirus vectors, poxvirus vectors, adeno-associated virus vectors, herpes simplex virus vectors, lentivirus vectors, baculovirus vectors, sendai virus vectors, measles virus vectors, simian virus vectors.
4. The cell of claim 3, wherein the viral vector is a vaccinia viral vector.
5. The cell of claim 1, wherein the cell is selected from the group consisting of: t cells, B cells, NK cells, neutrophils, monocytes, macrophages.
6. The cell of claim 1, wherein the immune cell is a T cell.
7. The cell of claim 6, wherein the immune cell is a T cell transduced with a T cell receptor.
8. The cell of claim 1, wherein the cell lacks endogenous expression of TRAC and/or 4-1BB protein.
9. A pharmaceutical composition comprising a cell according to any one of claims 1-8 and a pharmaceutically acceptable carrier.
10. Use of a cell according to any one of claims 1 to 8 or a pharmaceutical composition according to claim 9 for the preparation of a medicament for inhibiting a host-versus-graft reaction.
11. Use of a cell according to any one of claims 1 to 8 or a pharmaceutical composition according to claim 9 for the manufacture of a medicament for the treatment of cancer; the cancer is leukemia, lymphoma or multiple myeloma.
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Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103965361A (en) * 2013-02-06 2014-08-06 上海细胞治疗工程技术研究中心有限公司 Single chimeric converter for T-cell signal and application thereof
WO2014180288A1 (en) * 2013-05-06 2014-11-13 中国药科大学 Fusion protein having dual-functions for inhibiting angiogenesis in tumour microenvironment and activating adaptive immune response and gene and use thereof
CN104177499A (en) * 2013-05-27 2014-12-03 张鸿声 Chimeric antigen receptor, coding gene, expression vector and application thereof
CN107723275A (en) * 2017-10-20 2018-02-23 重庆精准生物技术有限公司 Universal CAR T cells and its preparation method and application
WO2019129056A1 (en) * 2017-12-28 2019-07-04 上海细胞治疗研究院 Cd137 bidirectional activation costimulatory molecule receptor and use thereof
CN109970864A (en) * 2017-12-28 2019-07-05 上海细胞治疗研究院 A bidirectionally activated costimulatory molecule receptor and use thereof
CN110272493A (en) * 2019-06-05 2019-09-24 南京凯地生物科技有限公司 Target specific chimeric antigen receptor T cell of CD19 and preparation method thereof and clinical application
CN113383071A (en) * 2018-11-01 2021-09-10 亘喜生物科技（上海）有限公司 Compositions and methods for T cell engineering
CN113372434A (en) * 2014-11-14 2021-09-10 豪夫迈·罗氏有限公司 Antigen binding molecules comprising TNF family ligand trimers

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103965361A (en) * 2013-02-06 2014-08-06 上海细胞治疗工程技术研究中心有限公司 Single chimeric converter for T-cell signal and application thereof
WO2014180288A1 (en) * 2013-05-06 2014-11-13 中国药科大学 Fusion protein having dual-functions for inhibiting angiogenesis in tumour microenvironment and activating adaptive immune response and gene and use thereof
CN104177499A (en) * 2013-05-27 2014-12-03 张鸿声 Chimeric antigen receptor, coding gene, expression vector and application thereof
CN113372434A (en) * 2014-11-14 2021-09-10 豪夫迈·罗氏有限公司 Antigen binding molecules comprising TNF family ligand trimers
CN107723275A (en) * 2017-10-20 2018-02-23 重庆精准生物技术有限公司 Universal CAR T cells and its preparation method and application
WO2019129056A1 (en) * 2017-12-28 2019-07-04 上海细胞治疗研究院 Cd137 bidirectional activation costimulatory molecule receptor and use thereof
CN109970864A (en) * 2017-12-28 2019-07-05 上海细胞治疗研究院 A bidirectionally activated costimulatory molecule receptor and use thereof
CN109970865A (en) * 2017-12-28 2019-07-05 上海细胞治疗研究院 A kind of CD137 bidirectionally activated costimulatory molecule receptor and its use
CN113383071A (en) * 2018-11-01 2021-09-10 亘喜生物科技（上海）有限公司 Compositions and methods for T cell engineering
CN110272493A (en) * 2019-06-05 2019-09-24 南京凯地生物科技有限公司 Target specific chimeric antigen receptor T cell of CD19 and preparation method thereof and clinical application

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
In Vivo Persistence, Tumor Localization, and Antitumor Activity of CAR-Engineered T Cells Is Enhanced by Costimulatory Signaling through CD137 (4-1BB);De-Gang Song等;Cancer Research;第71卷(第13期);4617-4627 *
嵌合抗原受体-T细胞免疫治疗在血液系统恶性肿瘤中的应用进展;克晓燕;;中国全科医学(第12期);6-11 *

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