TCR55 mutant and application thereof

Abstract

The invention relates to the field of biological medicines, in particular to a TCR55 mutant and application thereof.The TCR55 mutant comprises an alpha chain and a beta chain, the TCR55 mutant is obtained after mutation is conducted on the basis of wild TCR55, and the alpha chain of the TCR55 mutant is a mutant obtained after any one of the following sites on the alpha chain of the wild TCR55 is mutated into histidine, the fourth position D and / or the fifth position E of the CDR2 alpha, and the seventh position A of the CDR3 alpha; or the beta chain of the TCR55 mutant is a mutant obtained by mutating any one of the following on the beta chain of the wild type TCR55 into histidine: the second site N of CDR1beta, the second site A of CDR2beta, the fourth site E and / or the sixth site T of CDR2beta, and the thirteenth site Y of CDR3beta. The mutant can be used for identifying HIV (Pol448-456) / HLA-B35, has high activating power, triggers downstream signal transduction, and can be used for transforming T cells to form specific high-activating-power TCR-T cells for T cell adoptive immunotherapy.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceuticals, and in particular to a TCR55 mutant and its uses. Background Technology

[0002] T cells play a central role in cell-mediated immunity, recognizing and targeting cells infected with pathogens or cancer cells. The T cell receptor (TCR) is a membrane protein on the surface of T cells that recognizes short peptide antigens on the surface of corresponding target cells. In the immune system, the binding of the antigen-peptide-specific TCR to the short peptide-master histocompatibility complex (pMHC complex) initiates direct physical contact between T cells and antigen-presenting cells (APCs), triggering a series of cellular signaling and other physiological responses. This allows T cells with different antigen specificities to exert their immune effects on their target cells.

[0003] T-cell adoptive immunotherapy involves transferring T cells that specifically respond to target cell antigens into a patient, enabling them to exert their effects against the target antigens and target cells. However, the highly variable affinity of TCR-T cells isolated from each patient or donor limits their antitumor or antiviral efficacy in clinical trials. Furthermore, most antigen-specific mutations that trigger immune responses are found only in the individual's cancer, rather than in multiple patients, making generalization difficult.

[0004] Engineered antigen-specific TCR-T cells can overcome some of the challenges faced by current adoptive lymphocyte therapy because they can rapidly generate immunoreactive T lymphocytes with defined antigen specificity. There is a need in this field for TCRs that target different tumor antigens and provide high in vivo anti-tumor efficacy and low off-target effects. Common problems with TCR-T cell therapy include serious adverse events in clinical trials (such as central nervous system toxicity), which may be related to inappropriate target selection (so-called on-target / off-tumor effect) and biased expansion of the T cell population.

[0005] HIV, also known as human immunodeficiency virus, destroys CD4 in the human body. +If left uncontrolled, T lymphocytes will gradually eliminate the host's immune response over time. IPLTEEAEL (Pol448-456) is a short peptide derived from HIV antigens that can be recognized and presented by the HLC1 (HLA-B35) molecule on APC cells, forming an HIV (Pol448-456) / HLA-B35 pMHC complex. It has been reported that TCR55 can bind to HIV (Pol448-456) / HLA-B35, but this binding does not initiate T cell proliferation and activation, nor does it trigger downstream activation signals or T cell effector functions, including cytokine secretion and target cell killing. Therefore, finding TCR ligands with high activation capacity against HIV (Pol448-456) / HLA-B35 and engineering corresponding TCR-T cells holds promise for developing T cell adoptive immunotherapy with high anti-HIV efficacy and low off-target effects. Summary of the Invention

[0006] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a TCR55 mutant and its uses to solve the problems in the prior art.

[0007] To achieve the above and other related objectives, the present invention provides a TCR55 mutant comprising an α chain and a β chain. The α chain comprises CDR1α, CDR2α, and CDR3α, and the β chain comprises CDR1β, CDR2β, and CDR3β. The TCR55 mutant is obtained by mutating wild-type TCR55. The amino acid sequences of CDR1α in the α chain of the wild-type TCR55 are shown in SEQ ID NO.3, CDR2α in SEQ ID NO.4, and CDR3α in SEQ ID NO.5. The amino acid sequences of CDR1β in the β chain are shown in SEQ ID NO.6, CDR2β in SEQ ID NO.7, and CDR3β in SEQ ID NO.8. As shown in NO.8; the α chain of the TCR55 mutant is a mutant obtained by mutating one or more of the following sites on the α chain of wild-type TCR55 to histidine: Y at position 7 of CDR1α, D at position 4 and / or E at position 5 of CDR2α, and A at position 7 of CDR3α; or, the β chain of the TCR55 mutant is a mutant obtained by mutating one or more of the following sites on the β chain of wild-type TCR55 to histidine: N at position 2 of CDR1β, A at position 2, E at position 4 and / or T at position 6 of CDR2β, and Y at position 13 of CDR3β.

[0008] The present invention also provides an isolated polynucleotide encoding the TCR55 mutant.

[0009] The present invention also provides a nucleic acid construct containing the isolated polynucleotides described above.

[0010] The present invention also provides a lentiviral vector system, characterized in that the lentiviral vector system includes the aforementioned nucleic acid construct and auxiliary plasmid.

[0011] The present invention also provides a lentivirus, which is formed by viral packaging of the lentivirus vector system. The lentivirus contains the aforementioned nucleic acid construct.

[0012] The present invention also provides an engineered cell containing the aforementioned nucleic acid construct or containing the aforementioned lentivirus or having an exogenous polynucleotide integrated into its genome or expressing the aforementioned TCR55 mutant.

[0013] The present invention also provides the use of the TCR55 mutant, isolated polynucleotides, nucleic acid constructs, lentiviruses, engineered cells in the preparation of therapeutic drugs or in the preparation of diagnostic drugs.

[0014] As described above, the engineered highly activated TCR mutant that recognizes HIV antigens and its uses in this invention have the following beneficial effects: it can be used to recognize HIV (Pol448-456) / HLA-B35 and has high activation capacity, triggering downstream signal transduction, and can be used to modify T cells to form specific highly activated TCR-T cells for T cell adoptive immunotherapy. Attached Figure Description

[0015] Figure 1 The diagram shows a schematic of the modification method for the TCR mutant of the present invention.

[0016] Figure 2 The results show the positive rate of TCR55 expression in SKW3 cells.

[0017] Figure 3-1 The results show the activation effect of the TCRα mutant on different concentrations of HIV (Pol448-456).

[0018] Figure 3-2 The results show the activation effect of the TCRα mutant on different concentrations of HIV (Pol448-456).

[0019] Figure 4-1 The results show the activation effect of the TCRβ mutant on different concentrations of HIV (Pol448-456).

[0020] Figure 4-2The results show the activation effect of the TCRβ mutant on different concentrations of HIV (Pol448-456).

[0021] Figure 5 This shows the effect of TCRα mutant engineered T cells on downstream signal phosphorylation.

[0022] Figure 6 This shows the effect of TCRβ mutant engineered T cells on downstream signal phosphorylation. Detailed Implementation

[0023] This invention provides a TCR55 mutant comprising an α chain and a β chain. The α chain comprises CDR1α, CDR2α, and CDR3α, and the β chain comprises CDR1β, CDR2β, and CDR3β. The TCR55 mutant is obtained by mutating wild-type TCR55. The amino acid sequences of CDR1α in the α chain of the wild-type TCR55 are shown in SEQ ID NO.3, CDR2α in SEQ ID NO.4, and CDR3α in SEQ ID NO.5. The amino acid sequences of CDR1β in the β chain are shown in SEQ ID NO.6, CDR2β in SEQ ID NO.7, and CDR3β in SEQ ID NO.8. As shown in NO.8; the α chain of the TCR55 mutant is wild-type or obtained by mutating any one or more of the following sites on the α chain of wild-type TCR55 to histidine: Y at position 7 of CDR1α, D at position 4 and / or E at position 5 of CDR2α, A at position 7 of CDR3α; or, the β chain of the TCR55 mutant is wild-type or obtained by mutating any one or more of the following sites on the β chain of wild-type TCR55 to histidine: N at position 2 of CDR1β, A at position 2 of CDR2β, E at position 4 and / or T at position 6 of CDR2β, Y at position 13 of CDR3β.

[0024] In some embodiments of the present invention, the CDR of the α chain of the TCR55 mutant is wild-type, and the CDR of the β chain is obtained by mutating the CDR of the β chain of wild-type TCR55.

[0025] In some embodiments of the present invention, the number of mutation sites included in the CDR of the β chain of the TCR55 mutant is 1 to 5, for example, 1, 2, 3, 4 or 5.

[0026] In some embodiments of the present invention, the CDR and CDR2β, CDR3β of the α chain of the TCR55 mutant are wild type, and CDR1β is obtained by mutating the second N of CDR1β of wild type TCR55 to histidine.

[0027] In some embodiments of the present invention, the CDR and CDR1β, CDR3β of the α chain of the TCR55 mutant are wild-type, and CDR2β is obtained by mutating the second A of CDR2β of wild-type TCR55 to histidine.

[0028] In some embodiments of the present invention, the CDR and CDR1β, CDR3β of the α chain of the TCR55 mutant are wild-type, and CDR2β is obtained by mutating the 4th E of CDR2β of wild-type TCR55 to histidine.

[0029] In some embodiments of the present invention, the CDR and CDR1β, CDR3β of the α chain of the TCR55 mutant are wild-type, and CDR2β is obtained by mutating the 6th T of CDR2β of wild-type TCR55 to histidine.

[0030] In some embodiments of the present invention, the CDR and CDR1β, CDR2β of the α chain of the TCR55 mutant are wild type, and CDR3β is obtained by mutating the 13th Y position on CDR3β of wild type TCR55 to histidine.

[0031] Transfecting SKW-3 cells with mutants that mutated at any of the five sites above the β chain enhanced the proliferation and activation of TCR-T cells and initiated the activation of downstream signals.

[0032] In some embodiments of the present invention, the CDR of the α chain of the TCR55 mutant is obtained by mutating the α chain of wild-type TCR55 as described above, and the CDR of the β chain is wild-type.

[0033] In some embodiments of the present invention, the number of mutation sites included in the CDR of the α chain of the TCR55 mutant is 1 to 4, for example, 1, 2, 3, or 4.

[0034] In some embodiments of the present invention, in the α chain of the TCR55 mutant, CDR1α is formed by mutating the 7th Y position of CDR1α of wild-type TCR55 to histidine, and CDR2α, CDR3α and CDR of the β chain are wild-type.

[0035] In some embodiments of the present invention, in the α chain of the TCR55 mutant, CDR2α is formed by mutating the 4th D position of CDR2α in wild-type TCR55 to histidine, and CDR1α, CDR3α and CDR of the β chain are wild-type.

[0036] In some embodiments of the present invention, in the α chain of the TCR55 mutant, CDR2α is formed by mutating the 5th E position of CDR2α in wild-type TCR55 to histidine, and CDR1α, CDR3α and CDR of the β chain are wild-type.

[0037] In some embodiments of the present invention, CDR3α in the α chain of the TCR55 mutant is formed by mutating the 7th A position of CDR3α in wild-type TCR55 to histidine, and CDR1α, CDR2α and CDR of the β chain are wild-type.

[0038] Replacing any one of the four sites in the α chain with H can enhance the activation ability of wild-type TCR55 on HIV (Pol448-456) / HLA-B35 target antigens, triggering downstream signal transduction.

[0039] In some embodiments of the present invention, the α and β chains of the TCR55 mutant further include a framework region, which may be located between complementarity-determining regions (CDRs) or at both ends of the CDRs. The CDRs and the framework region combine to form a variable region. In some specific embodiments of the present invention, the sequence of the framework region is a variable region of a human TCR, or a framework region sequence of a mouse TCR variable region obtained by substitution, deletion, or addition of one or more amino acids (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3). This framework region sequence may have 80%, 85%, 90%, 93%, 95%, 97%, or 99% or more homology with the framework region sequence of the human TCR variable region.

[0040] In some embodiments of the present invention, the amino acid sequences of the variable regions of the α chain and β chain of the wild-type TCR55 are shown in SEQ ID NO.1 and 2, respectively.

[0041] TCR55α chain variable region sequence:

[0042] MLFSSLLCVFVAFSYSGSSVAQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGEGGAQKLVFGQGTRLTINPN(SEQ IDNO.1)

[0043] TCR55β chain variable region sequence:

[0044] MSIGLLCCVAFSLLWASPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHNSMYWYRQDPGMGLRLIYYSASEGTTDKGEVPNGYNVSRLNKREFSLRLESAAPSQTSVYFCASRTRGGTLIEQYFPGGTRLTVTE(SEQ IDNO.2)

[0045] In some embodiments of the present invention, the TCR55 mutant further includes a constant region.

[0046] In some embodiments of the present invention, the constant region amino acid sequences of the α and β chains of the TCR55 mutant are shown in SEQ ID NO. 27 and 28.

[0047] α-chain constant region sequence:

[0048] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS(SEQID NO.27)

[0049] β-chain constant region sequence:

[0050] DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG(SEQ ID NO.28)

[0051] The present invention also provides an isolated polynucleotide encoding the TCR55 mutant.

[0052] The nucleotide sequence of the isolated polynucleotide is not unique, as long as it can encode the TCR mutant. Those skilled in the art can deduce the corresponding nucleotide sequence based on the amino acid sequence of the TCR mutant.

[0053] The present invention also provides a nucleic acid construct containing the isolated polynucleotides described above.

[0054] The term "nucleic acid construct" refers to an artificially constructed nucleic acid segment that can be introduced into target cells or tissues. The nucleic acid construct can be various expression vectors, which include a vector backbone (empty vector) and an expression frame. The term "expression frame" refers to a sequence with the potential to encode a protein.

[0055] There is no specific limitation on the type of expression vector. An expression vector is a nucleic acid molecule that allows the insertion of foreign nucleotides without disrupting its ability to replicate and / or integrate into the host cell. Expression vectors may include nucleic acid sequences that allow them to replicate in the host cell, such as origins of replication. Expression vectors may also include one or more selective marker genes and other genetic factors. An expression vector is a vector containing the necessary regulatory sequences to enable the transcription and translation of one or more inserted genes. Expression vectors are selected from eukaryotic expression vectors or prokaryotic expression vectors.

[0056] The prokaryotic expression vector is selected from Escherichia coli expression vectors, Bacillus subtilis expression vectors, or Streptomyces expression vectors. In a preferred embodiment, the prokaryotic expression vector is selected from Escherichia coli expression vectors.

[0057] The eukaryotic expression vector is selected from yeast expression vectors, insect expression vectors, or mammalian expression vectors. The mammalian expression vector is either a non-viral expression vector or a viral expression vector. The viral expression vector is selected from retroviral expression vectors, lentiviral expression vectors, adenovirus expression vectors, and adeno-associated virus expression vectors.

[0058] The host cells are selected from eukaryotic or prokaryotic host cells. Eukaryotic host cells are selected from fungi such as yeast, insects, birds, plants, *C. elegans* or nematodes, or mammalian host cells. A non-limiting example of insect cells is *Noctua przewalskii* cells. Examples of yeast host cells are *Saccharomyces cerevisiae*, *Kluyveromyces lactis*, or *Yersinia lipolytica*. Examples of mammalian cells are COS cells, juvenile hamster kidney cells, mouse L cells, LNCaP cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, African green monkey cells, CV1 cells, Vero, or Hep-2 cells. Examples of prokaryotic host cells include bacterial cells such as *Escherichia coli*, *Streptomyces*, *Bacillus subtilis*, *Salmonella typhi*, or mycobacteria.

[0059] Those skilled in the art can transfect the expression vector into host cells using methods well-known in the art to obtain cells containing the encoding gene of the antibody of the present invention. For example, the expression vector can be introduced into eukaryotic cells via liposome transfection.

[0060] The present invention also provides a lentiviral vector system, characterized in that the lentiviral vector system includes the aforementioned nucleic acid construct and auxiliary plasmid.

[0061] Furthermore, the helper plasmid encodes one or more nucleotide sequences of the gag, pol, Rev, and VSVg proteins, as well as nucleotide sequences of other essential viral packaging components. All helper plasmids are commercially available.

[0062] Furthermore, the lentiviral vector system also includes a host cell, which can be a cell that produces lentiviruses, such as a mammalian cell, specifically a 293T cell or a cell derived therefrom.

[0063] The lentivirus can be obtained by transfecting host cells using the nucleic acid construct and helper plasmid in the lentiviral vector system. The host cell can be a mammalian cell.

[0064] The present invention also provides a lentivirus, which is formed by viral packaging of the lentivirus vector system. The lentivirus contains the aforementioned nucleic acid construct.

[0065] The present invention also provides an engineered cell containing the aforementioned nucleic acid construct or containing the aforementioned lentivirus or having an exogenous polynucleotide integrated into its genome or expressing the aforementioned TCR55 mutant.

[0066] The engineered cells are T cells. The T cells are primary T cells or passaged T cells. For example, the passaged T cells are T cells derived from cancer patients.

[0067] The present invention also provides an antibody drug comprising the aforementioned TCR55 mutant.

[0068] In some specific embodiments, the antibody drug may be a TCR-BiTE (Bispecific T-cell Engagers) antibody protein drug.

[0069] Furthermore, the TCR-BiTE antibody protein drug includes the aforementioned TCR55 mutant, and also includes a single-chain variable fragment (scFv) against any one of CD3, CD4, CD5, CD6, CD7, CD8, CD28 or 4-1BB.

[0070] The present invention also provides the use of the TCR55 mutant, isolated polynucleotide, nucleic acid construct, lentivirus, engineered cell or antibody drug in the preparation of therapeutic drugs or in the preparation of diagnostic drugs.

[0071] In some embodiments of the present invention, the therapeutic agent is an anti-tumor drug or a drug for treating viral infections such as HIV infection. The tumor is selected from melanoma, leukemia, synovial sarcoma, multiple myeloma, lung cancer, liver cancer, cervical cancer, pancreatic cancer, ovarian cancer, etc.

[0072] This invention also proposes a method for detecting recombinant cell activation. This method utilizes recombinant T cells engineered with TCR55 mutants, co-cultures them with APCs presenting HIV (Pol448-456) / HLA-B35, and then detects the activation status of downstream T cells.

[0073] The present invention also provides a treatment for the disease, the treatment comprising administering the engineered cells to a subject in need.

[0074] The disease is cancer or a viral infection such as HIV infection. The treatment method is T-cell adoptive immunotherapy.

[0075] The cancers mentioned are selected from melanoma, leukemia, synovial sarcoma, multiple myeloma, lung cancer, liver cancer, cervical cancer, pancreatic cancer, ovarian cancer, etc.

[0076] In this application, "tumor" or "cancer" refers to any medical condition mediated by the growth, proliferation, or metastasis of tumor or malignant cells, resulting in solid tumors and non-solid tumors such as leukemia. In this invention, "tumor" refers to the solid matter of tumors and / or malignant cells.

[0077] "Subjects" include, but are not limited to, animals, preferably mammals; said mammals are preferably rodents, even-toed ungulates, perissodactyls, lagomorphs, primates, etc. The mammals include, for example, humans, non-human primates (e.g., monkeys), mice, pigs, cattle, goats, rabbits, rats, guinea pigs, hamsters, horses, monkeys, sheep, or other non-human mammals; non-mammals include, for example, non-mammal vertebrates, such as birds (e.g., chickens or ducks) or fish, and non-mammal invertebrates. Subjects can be humans, such as patients with weakened immune systems or cancer.

[0078] "Treatment" or "therapy" for a condition includes preventing or alleviating the condition, slowing the onset or progression of the condition, reducing the risk of developing the condition, preventing or delaying the development of symptoms associated with the condition, reducing or terminating symptoms associated with the condition, achieving complete or partial reversal of the condition, curing the condition, or a combination of the above. For cancer, "treatment" or "therapy" can refer to inhibiting or slowing the growth, proliferation, or metastasis of tumors or malignant cells, or some combination of the above. For tumors, "treatment" or "therapy" includes eliminating all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying tumor development, or some combination of the above.

[0079] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0080] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0081] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0082] Example 1: CDR region analysis of TCR55 and HIV (Pol448-456) / HLA-B35 complex

[0083] In existing reports, although the HIV (Pol448-456) / HLA-B35 antigenic peptide specifically recognizes the ligand TCR55, the binding of TCR55 to HIV (Pol448-456) / HLA-B35 does not activate T cell activation, nor does it initiate downstream activation signals to induce an immune response. Based on this, this embodiment proposes several TCR55 mutants, which are amino acid mutations occurring in the wild-type amino acid sequence of TCR55 as shown below.

[0084] TCRα:

[0085] MLFSSLLCVFVAFSYSGSSVAQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGEGGAQKLVFGQGTRLTINPN(SEQ IDNO.1)

[0086] TCRβ:

[0087] MSIGLLCCVAFSLLWASPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHNSMYWYRQDPGMGLRLIYYSASEGTTDKGEVPNGYNVSRLNKREFSLRLESAAPSQTSVYFCASRTRGGTLIEQYFPGGTRLTVTE(SEQ IDNO.2)

[0088] This invention involves sequentially histidine-mutating the amino acid sequence of the CDR region to identify mutant TCRs with higher activation capacity compared to wild-type TCR55. The CDR region sequences are shown in the table below:

[0089] Table 1. CDR region of TCRα

[0090]

[0091] Table 2. CDR region of TCRβ

[0092]

[0093]

[0094] Example 2: Construction of recombinant plasmids for TCR55 CDR region mutants

[0095] 2.1 Construction of CDR region mutants of TCR55

[0096] In this embodiment, TCR55 was used as the parental template for the synthesis and recombinant expression of the template pHR-TCR55. Specifically: the TCR55 gene sequence was synthesized into the pHR vector (addgene, 79121) and named pHR-TCR55; the plasmid pHR-TCR55 was linearized using MluI and NotI restriction enzymes, and the linearization product was detected by 1% agarose gel electrophoresis and then recovered. Site-directed amino acid mutagenesis was performed using conventional overlap extension PCR. Using the pHR-TCR55 plasmid as a template, mutant primers were designed to replace each amino acid with H in the CDR region of TCRα and TCRβ, with the mutated base placed in the middle of the primer. The sequences of some mutants and the upstream and downstream mutant primers used in this embodiment are shown in Table 3 below. The mutation sites in the table are the sites in the sequences shown in SEQ ID NO. 1 and 2.

[0097] Table 3 Sequences of mutants and upstream / downstream mutant primers

[0098]

[0099] PCR amplification system (50 μL): pHR-TCR55 1 μL (50 ng), corresponding upstream and downstream mutant primers 1 μL each (10 μM), 5×Pfu Buffer 10 μL, Pfu enzyme 1 μL, and finally add sterile distilled water to make up the volume to 50 μL. PCR reaction program: (1) 98℃ pre-denaturation for 2 min; (2) 98℃ denaturation for 20 s; (3) 55℃ annealing for 30 s; (4) 72℃ extension for 1 min, denaturation-annealing-extension for a total of 30 cycles, and finally 72℃ extension for 1 min, and store the PCR product at 4℃.

[0100] 2.2 Synthesis of TCR55 mutant recombinant plasmid

[0101] The nucleotide sequence encoding the mutant fragment was inserted into the linearized pHR vector using an in-fusion method. The vector was then transformed into *E. coli* DH5α competent cells using a heat shock method, and positive transformants were screened on LB agar (containing 100 μg / mL ampicillin, Amp). The next day, single clones were picked for colony PCR; the PCR amplification system (10 μL) consisted of: 1 μL of single-clone bacterial culture, 0.2 μL each of primers PF and PR, 5 μL of 2×Taq DNA polymerase mix (containing buffer), and finally, sterile distilled water was added to bring the volume to 10 μL. The sequences of primers PF and PR are shown in Table 4 below.

[0102] Table 4. Sequences of primers PF and PR

[0103] Primers sequence Serial Number PF AGCTCACAACCCCTCACTC SEQ ID NO.29 PR CATAGCGTAAAAGGAGCAACA SEQ ID NO.30

[0104] The PCR reaction procedure was as follows: (1) 95℃ pre-denaturation for 3 min; (2) 95℃ denaturation for 20 s; (3) 55℃ annealing for 30 s; (4) 72℃ extension for 1 min, and the denaturation-annealing-extension cycle was repeated for 35 cycles. Finally, the extension was performed at 72℃ for 5 min. The PCR products were detected by 1% agarose gel electrophoresis, and the positive transformants were sent for sequencing. The sequencing results were compared with the gene sequence of wild-type pHR-TCR55 using SnapGene software, and plasmids were extracted from the correctly sequenced strains.

[0105] Example 3: Preparation of recombinant TCR55-T cells and screening of highly activated TCR55 mutants

[0106] 3.1 Lentiviral Packaging of TCR55 Mutants

[0107] HEK293T-derived LentiX cells were used as host cells for lentivirus packaging to produce viral proteins at high levels. On day 1, LentiX cells were loaded at 3 × 10⁻⁶ cells per cell line. 5 Cells / mL were seeded into 6-well plates (2 mL per well), i.e., 6 × 10⁶ cells per well. 5On the second day, for each TCR, 750 ng of recombinant plasmid, 500 ng of psPAX (Addgene, 12260), and 250 ng of pMD2.G (Addgene, 12259) were added to 100 μL of opti-MEM medium, mixed well, and then 4.5 μL of PEI was added. The mixture was gently mixed several times with a pipette and incubated at room temperature for 20 min. During this period, the old medium in each well was replaced with fresh cRPMI (RPMI 1640 medium + 10% FBS + 1× penicillin / streptomycin) medium. Finally, the DNA / PEI / opti-MEM mixture was slowly added dropwise to each well and the cells were incubated at 37°C and 5% CO2 for 48 h. 48 h after transfection, the virus was collected in 15 mL centrifuge tubes, centrifuged at 500 g and 4°C for 5 min to remove cell debris and impurities, and then filtered through a 0.45 μM filter membrane. The filtered viral supernatant was collected for infection.

[0108] 3.2 Preparation of recombinant TCR55-T cells by infecting SKW-3 cells with TCR55 mutant virus

[0109] SKW-3 cells were administered at a rate of 0.3 × 10⁻⁶. 6 Cells were cultured at a density of 37°C and 5% CO2 for 2 days before being used for infection with the TCR55 mutant virus. The concentration of SKW-3 cells was adjusted to 1×10⁻⁶ cells / mL for cell counting. 6 Working cell suspension per mL. Infection was performed using lentiviral suspensions of TCR55 wild-type TCRα and its mutants or wild-type TCRβ and its mutants. Specifically, 1 mL of working cell suspension was added to a 6-well plate, followed by 2 mL of TCRα virus solution and 2 mL of TCRβ virus solution. After gentle mixing, the plate was incubated at 37°C and 5% CO2 for 2 days. Three different infection types were designed for this study: ① 2 mL wild-type TCRα + 2 mL wild-type TCRβ; ② 2 mL mutant TCRα + 2 mL wild-type TCRβ, where the TCRα mutant included the aforementioned four mutant types; ③ 2 mL wild-type TCRα + 2 mL mutant TCRβ, where the TCRβ mutant included the aforementioned five mutant types.

[0110] like Figure 2 As shown, after infection, cells were stained with anti-human TCRα / β (APC) antibody and analyzed by flow cytometry. It was found that the infection efficiency of TCR55 wild-type and its mutants could reach more than 80% compared with the negative control (uninfected SKW3 cells).

[0111] Example 4: Screening for highly activated TCR55 mutants

[0112] The activation of the HIV (Pol448-456) antigenic peptide by TCR55 mutants was assessed by stimulating recombinant TCR55-SKW3 cells with HIV (Pol448-456)-loaded APC cells. In this embodiment, the T cell activation surface marker CD69 was used to indicate the T cell proliferation activation response to the antigenic peptide. Specific method: 10 mg of HIV (Pol448-456) antigenic peptide was dissolved in 100 μL DMSO to obtain a 100 mM antigenic peptide stock solution. The concentration of KG-1 cells was adjusted to 5 × 10⁻⁶ cells / mL. 5 Cells / mL. Add 100 μL of KG-1 cell suspension to each well of a U-shaped 96-well plate, and simultaneously add an appropriate volume of antigen peptide solution to each well to achieve an antigen peptide concentration of 10. -5 ~10 -11 M was used to prepare APC cells. Finally, KG-1 cells were gently mixed with peptide and cultured at 37°C and 5% CO2 for 3 hours. After culture, the cells were centrifuged at 400g and 4°C for 5 minutes to remove excess peptides for later use. The concentration of recombinant TCR55-SKW3 cells was adjusted to 5 × 10⁻⁶ cells / mL using fresh cRPMI medium. 5 Cells / mL. 100 μL of different types of TCR55-SKW3 cells were added to KG-1-peptide cell culture plates and co-incubated at 37℃ and 5% CO2 for 14 h. After co-incubation, the cell culture plates were removed at 400 g and centrifuged at 4℃ for 5 min. The cells were stained with anti-CD69-APC (1:500) and anti-αβTCR-BV421 (1:500) on ice for 30 min, and the activation of HIV (Pol448-456) by different types of TCR mutants was analyzed by flow cytometry. Results are as follows: Figure 3-1 As shown, compared with wild-type TCRα, the TCRα chain mutants Y33H, D54H, E55H, and A98H enhanced the T activation ability of recombinant T cells against the HIV (Pol448-456) antigen peptide to varying degrees. Similarly, the TCRα chain mutants Y33H, D54H, E55H, and A98H... Figure 4-1 It was found that, compared to wild-type TCRβ recombinant T cells, TCR55β chain mutants N28H, A50H, E52H, and T54H all significantly increased T cell activation capacity. Other mutants (such as...) Figure 3-2 and Figure 4-2 It cannot increase the activation capacity of T cells.

[0113] Example 5: Effects of TCR55 mutant on downstream phosphorylation signaling in T cells

[0114] TCR activation initiates the recruitment and phosphorylation of downstream adaptor or cytoskeletal proteins, thus the transmission of downstream signals of the immune response can be indicated by phosphorylated ERK staining. In this example, eleven engineered recombinant TCR-SKW3 cells were used as test materials: TCR55 composed of four mutants of TCRα (wild-type and α-chain Y33H, D54H, E55H, A98H) and wild-type β-chain, and TCR55 composed of TCRβ (wild-type and β-chain N28H, A50H, E52H, T54H, Y104H) and wild-type β-chain. APC cell preparation was as described in Example 4. APC cells and engineered recombinant SKW-3 cells were co-incubated at 37°C and 5% CO2 for 15 min. After incubation, the recombinant SKW-3 cells were immediately fixed with 4% PFA for 15 min. Then, they were washed once with PBS (containing 0.5% BSA) and treated with pre-chilled methanol on ice for 30 min. After treatment, cells were washed twice with PBS (containing 0.5% BSA), and finally anti-pERK1 / 2 (1:50) antibody was added. The cells were incubated at room temperature with shaking for 1 hour. After staining, cells were washed once with PBS (containing 0.5% BSA) and then analyzed by flow cytometry. Results are as follows: Figure 5 and Figure 6 As shown, compared to wild-type TCRα recombinant T cells, mutations of Y at position 33, D at position 54, E at position 55, and A at position 98 in TCRα to H resulted in varying degrees of increase in downstream phosphorylation signals in T cells. Similarly, compared to wild-type TCRβ, mutants of the TCRβ chain N28H, A50H, E52H, T54H, and Y104H all significantly increased downstream phosphorylation signals, evoking a more activated immune response.

[0115] Example 6: Affinity Detection of TCR55 Mutant

[0116] The TCR-pMHC affinity was measured on a Biacore 8K instrument as follows: Biotinylated pMHC protein was immobilized onto a streptavidin chip until the response units (RU) reached 100-200 RU. The TCR protein was serially diluted and added to the pMHC chip, and the affinity was measured at room temperature.

[0117] The results are shown in the table below.

[0118] TCR <![CDATA[K D (μM)]]> TCR55α-Y33H 3.56 TCR55α-D54H 8.68 TCR55α-E55H 2.39 TCR55β-N28H 5.00 TCR55β-E52H 3.28 TCR55β-T54H 15.53 TCR55β-Y104H 0.54

[0119] In summary, this application uses the TCR55 amino acid sequence shown in the examples as a template, and modifies the CDR regions of the α and β chains of TCR55 based on this amino acid sequence to obtain a series of mutants with significantly enhanced activation ability against HIV (Pol448-456) / HLA-B35 antigen peptides. The T cells modified with the TCR55 mutants provided in this application can effectively respond to HIV (Pol448-456) antigen peptides and strongly activate downstream phosphonucleotide signals to induce an immune response, suggesting that this mutant has good application prospects in the treatment of HIV-infected populations.

[0120] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A TCR55 mutant, characterized in that, The TCR55 mutant comprises an α chain and a β chain. The α chain includes CDR1α, CDR2α, and CDR3α, and the β chain includes CDR1β, CDR2β, and CDR3β. The TCR55 mutant is obtained by mutating wild-type TCR55. The amino acid sequences of CDR1α in the α chain of the wild-type TCR55 are shown in SEQ ID NO.3, CDR2α in SEQ ID NO.4, and CDR3α in SEQ ID NO.

5. The amino acid sequences of CDR1β in the β chain are shown in SEQ ID NO.6, CDR2β in SEQ ID NO.7, and CDR3β in SEQ ID NO.

8. As shown in NO.8; the α chain of the TCR55 mutant is a mutant obtained by mutating one or more of the following sites on the α chain of wild-type TCR55 to histidine: Y at position 7 of CDR1α, D at position 4 and / or E at position 5 of CDR2α, and A at position 7 of CDR3α; or, the β chain of the TCR55 mutant is a mutant obtained by mutating one or more of the following sites on the β chain of wild-type TCR55 to histidine: N at position 2 of CDR1β, A at position 2, E at position 4 and / or T at position 6 of CDR2β, and Y at position 13 of CDR3β.

2. The TCR55 mutant according to claim 1, characterized in that, The CDR of the α chain of the TCR55 mutant is wild-type, and the CDR of the β chain is mutant; or, the CDR of the α chain is mutant, and the CDR of the β chain is wild-type.

3. The TCR55 mutant according to claim 1, characterized in that, The TCR55 mutant is selected from any of the following: 1) The CDR and CDR2β, CDR3β of the α chain of the TCR55 mutant are wild type, and CDR1β is obtained by mutating the second N of CDR1β of wild type TCR55 to histidine. 2) The CDR and CDR1β, CDR3β of the α chain of the TCR55 mutant are wild type, and CDR2β is obtained by mutating the second A of CDR2β of wild type TCR55 to histidine. 3) The CDR and CDR1β, CDR3β of the α chain of the TCR55 mutant are wild type, and CDR2β is obtained by mutating the 4th E of CDR2β of wild type TCR55 to histidine. 4) The CDR and CDR1β, CDR3β of the α chain of the TCR55 mutant are wild type, and CDR2β is obtained by mutating the 6th T of CDR2β of wild type TCR55 to histidine. 5) The CDR and CDR1β, CDR2β of the α chain of the TCR55 mutant are wild type, and CDR3β is obtained by mutating the 13th Y position of CDR3β of wild type TCR55 to histidine. 6) In the α chain of the TCR55 mutant, CDR1α is formed by mutating the 7th Y position of CDR1α in wild-type TCR55 to histidine, while CDR2α, CDR3α and CDR of the β chain are wild-type. 7) In the α chain of the TCR55 mutant, CDR2α is formed by mutating the 4th D position of CDR2α in wild-type TCR55 to histidine, while CDR1α, CDR3α and CDR of the β chain are wild-type. 8) In the α chain of the TCR55 mutant, CDR2α is formed by mutating the 5th E position of CDR2α in wild-type TCR55 to histidine, while CDR1α, CDR3α and CDR of the β chain are wild-type. 9) In the α chain of the TCR55 mutant, CDR3α is formed by mutating the 7th A position of CDR3α in wild-type TCR55 to histidine, while CDR1α, CDR2α and CDR of the β chain are wild-type.

4. The TCR55 mutant according to claim 1, characterized in that, The amino acid sequences of the variable regions of the α chain and β chain of the wild-type TCR55 are shown in SEQ ID NO.1 and 2, respectively.

5. The TCR55 mutant according to claim 1, characterized in that, The amino acid sequences of the α-chain and β-chain constant regions of the wild-type TCR55 are shown in SEQ ID NO.27 and 28, respectively.

6. An isolated polynucleotide, characterized in that, The isolated polynucleotide encodes the TCR55 mutant as described in any one of claims 1 to 5.

7. A nucleic acid construct, characterized in that, Contains the isolated polynucleotide as described in claim 6.

8. A lentivirus vector system, characterized in that, The lentiviral vector system includes the nucleic acid construct as described in claim 7 and an auxiliary plasmid.

9. A lentivirus, characterized in that, The lentivirus is formed by viral packaging of the lentivirus vector system as described in claim 8.

10. An engineered cell, characterized in that, The engineered cells contain the nucleic acid constructs described above, or contain the lentiviruses described above, or have the exogenous polynucleotides described above integrated into the genome, or express the TCR55 mutant.

11. The engineered cell according to claim 10, characterized in that, The engineered cells are TCR-T cells.

12. An antibody drug, characterized in that, The antibody drug comprises the TCR55 mutant as described in any one of claims 1-5.

13. The antibody drug according to claim 12, characterized in that, The antibody drug is a TCR-BiTE antibody protein drug.

14. Use of the TCR55 mutant of any one of claims 1 to 5, the isolated polynucleotide of claim 6, the nucleic acid construct of claim 7, the lentivirus of claim 9, the engineered cell of claim 10, or the antibody drug of claim 11 or 12 in the preparation of a therapeutic drug or in the preparation of a diagnostic drug.

15. The use according to claim 14, characterized in that, The therapeutic drug is a tumor treatment drug or a viral infection treatment drug; preferably, the tumor is selected from melanoma, leukemia, synovial sarcoma, multiple myeloma, lung cancer, liver cancer, cervical cancer, pancreatic cancer, and ovarian cancer; preferably, the viral infection treatment drug is an HIV infection treatment drug.

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