CD20 and CD19 targeting single-chain antibody and chimeric antigen receptor and application thereof

Abstract

The invention provides a CD20 and CD19 targeting single-chain antibody, a chimeric antigen receptor and application thereof, and relates to the technical field of biology. The CD20 and CD19 targeting single-chain antibody provided by the invention is obtained by screening of the inventor, and has higher CD19 and CD20 affinity and good stability. The chimeric antigen receptor targeting CD20 and CD19 provided by the invention has good stability, can improve the targeting ability of T cells to tumor cells after being expressed in the T cells, and is further used for tumor treatment.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a single-chain antibody and chimeric antigen receptor targeting CD20 and CD19 and their applications. Background Technology

[0002] In the field of lymphoma treatment, existing antibody drugs (such as rituximab targeting CD20 or bispecific antibodies targeting CD19) have the following technical shortcomings: Immunogenicity issues: Most existing antibodies are murine or chimeric. Even with partial humanization, residual non-human sequences can still trigger immune rejection (such as the HAMA reaction), leading to increased drug clearance or decreased efficacy. For example, clinical data shows that approximately 30% of patients develop anti-drug antibodies (ADA) after using chimeric antibodies.

[0003] Limitations of single-target: Single-target antibodies (such as those targeting only CD20 or CD19) are prone to treatment failure due to tumor antigen escape (such as CD19-negative clonal amplification) or antigen downregulation. For example, 30%-50% of patients who relapse after CD19-CAR-T therapy experience CD19 antigen loss.

[0004] Insufficient structural stability: Traditional antibodies have hinge regions (such as the IgG1 hinge) that are easily degraded by proteases, and their transmembrane regions are not optimized, resulting in short half-lives (about 7 days) or accumulation in vivo.

[0005] Low T cell activation efficiency: Existing bispecific antibodies (such as CD19 / CD3 BiTE) lack co-stimulatory signals (such as CD28 or 4-1BB) and rely solely on CD3ζ to activate T cells, leading to T cell exhaustion or insufficient proliferation.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The primary objective of this invention is to provide a single-chain antibody (scfv) that targets CD20 and CD19.

[0008] A second objective of this invention is to provide a chimeric antigen receptor that targets CD20 and CD19 to solve the aforementioned technical problems.

[0009] A third objective of this invention is to provide a nucleic acid.

[0010] The fourth objective of this invention is to provide a carrier.

[0011] The fifth objective of this invention is to provide a cell.

[0012] The sixth objective of this invention is to provide the use of the above-mentioned chimeric antigen receptor in the preparation of products for treating lymphoma.

[0013] To achieve the above objectives, the following technical solution is adopted: In a first aspect, the present invention provides a single-chain antibody targeting CD20 and CD19, the amino acid sequence of which is shown in SEQ ID NO.1.

[0014] In a second aspect, the present invention provides a chimeric antigen receptor that targets CD20 and CD19, wherein the chimeric antigen receptor comprises a single-chain antibody.

[0015] As a further technical solution, the chimeric antigen receptor comprises, from the N-terminus to the C-terminus, a signal peptide, the single-chain antibody, a CD8a hinge region, a CD8a transmembrane domain, a CD137 co-stimulatory receptor, and a CD3Z co-stimulatory receptor, which are sequentially connected in series.

[0016] As a further technical solution, the amino acid sequence of the signal peptide is shown in SEQ ID NO.2; The amino acid sequence of the CD8a hinge region is shown in SEQ ID NO.3; The amino acid sequence of the CD8a transmembrane domain is shown in SEQ ID NO.4; The amino acid sequence of the CD137 co-stimulatory receptor is shown in SEQ ID NO.5; The amino acid sequence of the CD3Z co-stimulatory receptor is shown in SEQ ID NO.6.

[0017] Thirdly, the present invention provides a nucleic acid that encodes the single-chain antibody or chimeric antigen receptor.

[0018] Fourthly, the present invention provides a carrier carrying the nucleic acid.

[0019] Fifthly, the present invention provides a cell that carries the nucleic acid, or contains the vector, or expresses the single-chain antibody or chimeric antigen receptor.

[0020] As a further technical solution, the cells include T cells or macrophages.

[0021] In a sixth aspect, the present invention provides the use of the above-mentioned chimeric antigen receptor in the preparation of products for treating lymphoma.

[0022] As a further technical solution, the product includes T cells; The T cells express the chimeric antigen receptor.

[0023] Compared with the prior art, the present invention has the following beneficial effects: The single-chain antibody targeting CD20 and CD19 provided by this invention was screened by the inventors and has higher affinity for CD19 and CD20, as well as good stability.

[0024] The chimeric antigen receptor targeting CD20 and CD19 provided by this invention has good stability. When expressed in T cells, it can enhance the targeting ability of T cells to tumor cells, and thus be used for tumor treatment. Attached Figure Description

[0025] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0026] Figure 1 For affinity testing data; Figure 2 To incorporate the strength test results; Figure 3 The results are for the competitive binding rate test. Figure 4 The results are for the monomer ratio and binding retention rate. Figure 5 This represents the cell death rate result. Detailed Implementation

[0027] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0028] The term "vector" refers to a nucleic acid delivery vehicle into which nucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material they carry to be expressed in the host cells.

[0029] The vectors described herein are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses. In some embodiments, the vectors of this invention contain regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0030] In a first aspect, the present invention provides a single-chain antibody targeting CD20 and CD19, the amino acid sequence of which is shown in SEQ ID NO.1: EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYFASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPCTFGQGTRLEIKGGGGSGGGGSEVQLVE SGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSRISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNCYYGMDVWGQGTTVTVSSGGGGSGG GGSGGGGSEVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAMHWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDYYYGMDVWGQGTTVTV SSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYQTSQLQSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPATFGQGTKVEIK (SEQ ID NO.1).

[0031] The single-chain antibody targeting CD20 and CD19 provided by this invention was screened by the inventors and has higher affinity for CD19 and CD20, as well as good stability.

[0032] In a second aspect, the present invention provides a chimeric antigen receptor that targets CD20 and CD19, wherein the chimeric antigen receptor comprises a single-chain antibody.

[0033] The chimeric antigen receptor targeting CD20 and CD19 provided by this invention has good stability. When expressed in T cells, it can enhance the targeting ability of T cells to tumor cells, and thus be used for tumor treatment.

[0034] In some alternative embodiments, the chimeric antigen receptor comprises, from the N-terminus to the C-terminus, a signal peptide, the single-chain antibody, a CD8a hinge region, a CD8a transmembrane domain, a CD137 co-stimulatory receptor, and a CD3Z co-stimulatory receptor, in tandem.

[0035] In some alternative embodiments, the amino acid sequence of the signal peptide is as shown in SEQ ID NO.2: MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO. 2).

[0036] The amino acid sequence of the CD8a hinge region is shown in SEQ ID NO.3: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO. 3).

[0037] The amino acid sequence of the CD8a transmembrane domain is shown in SEQ ID NO.4: IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO. 4).

[0038] The amino acid sequence of the CD137 co-stimulatory receptor is shown in SEQ ID NO.5: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO. 5).

[0039] The amino acid sequence of the CD3Z co-stimulatory receptor is shown in SEQ ID NO. 6: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO. 6).

[0040] Thirdly, the present invention provides a nucleic acid that encodes the single-chain antibody or chimeric antigen receptor.

[0041] Fourthly, the present invention provides a carrier carrying the nucleic acid.

[0042] Fifthly, the present invention provides a cell that carries the nucleic acid, or contains the vector, or expresses the single-chain antibody or chimeric antigen receptor.

[0043] In some alternative implementations, the cells include, but are not limited to, T cells or macrophages.

[0044] In a sixth aspect, the present invention provides the use of the above-mentioned chimeric antigen receptor in the preparation of products for treating lymphoma.

[0045] For example, expressing the antigen-chimeric receptor provided by this invention in T cells or macrophages can enhance the targeting ability of T cells or macrophages for lymphoma, thereby achieving the treatment of lymphoma.

[0046] In some alternative implementations, the product includes, but is not limited to, T cells; The T cells express the chimeric antigen receptor.

[0047] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0048] Example 1 A single-chain antibody targeting CD20 and CD19, the amino acid sequence of which is shown in SEQ ID NO.1.

[0049] Comparative Example 1 A single-chain antibody targeting CD20 and CD19, the amino acid sequence of which is shown in SEQ ID NO.7: EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSGGGGS GGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKRTGSTSGSGKP GSGEGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGG STKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS (SEQ ID NO.7).

[0050] Example 2 The chimeric antigen receptor targets CD20 and CD19. The chimeric antigen receptor consists of a signal peptide, a single-chain antibody, a CD8a hinge region, a CD8a transmembrane domain, a CD137 co-stimulatory receptor, and a CD3Z co-stimulatory receptor, which are sequentially linked from the N-terminus to the C-terminus.

[0051] The amino acid sequence of the signal peptide is shown in SEQ ID NO.2.

[0052] The amino acid sequence of the single-chain antibody is shown in SEQ ID NO.1.

[0053] The amino acid sequence of the CD8a hinge region is shown in SEQ ID NO.3.

[0054] The amino acid sequence of the CD8a transmembrane domain is shown in SEQ ID NO.4.

[0055] The amino acid sequence of the CD137 co-stimulatory receptor is shown in SEQ ID NO.5.

[0056] The amino acid sequence of the CD3Z co-stimulatory receptor is shown in SEQ ID NO.6.

[0057] Comparative Example 2 The chimeric antigen receptors targeting CD20 and CD19 differ from those in Example 1 in that the amino acid sequence of the single-chain antibody is shown in SEQ ID NO.7.

[0058] Experimental Example 1 The single-chain antibody of Example 1 of this invention was obtained by screening animal immune antibodies and humanizing the antibodies.

[0059] SPR experiment: CD19 / CD20 antigens were immobilized on the chip surface, and scFv solutions of different concentrations were flowed through them while binding and dissociation signals were monitored in real time. The affinity constant and dissociation rate were calculated. The results are as follows: Figure 1 As shown in the results, the CD19 and CD20 affinities (KD values) of the scFv in Example 1 (optimized) were significantly improved, reaching approximately 1 nM and 0.7 nM, respectively.

[0060] Flow cytometry binding assay: Serially diluted scFv was co-incubated with CD19+ / CD20+ tumor cells, labeled with fluorescent secondary antibody, and the binding strength was detected by flow cytometry to calculate the half-maximal effective concentration.

[0061] Flow cytometry combined experiment ( Figure 2 The results showed that the scFv of Example 1 (optimized) significantly reduced the EC50 values ​​of NALM-6, Raji and Daudi cells, and increased the binding efficiency by about 2-3 times.

[0062] Competitive binding assay: scFv was co-incubated with fluorescently labeled clinical antibodies in target cells, and the degree of fluorescence signal attenuation was detected by flow cytometry to quantify the competitive occupancy ability of scFv for antigenic epitopes.

[0063] In the competitive binding experiment, the antigen occupancy rate of scFv in Example 1 (optimized) increased to approximately 70%, significantly better than the 35-40% before optimization (Comparative Example 1). Figure 3 ).

[0064] Serum stability test: scFv was placed in human serum at 37°C to simulate the in vivo environment. Samples were taken periodically and its structural integrity and functional residual rate were analyzed by chromatography and activity detection.

[0065] Serum stability was significantly improved; after 72 hours, the monomeric proportion of scFv in Example 1 (optimized) remained above 85%, and the binding activity retention rate exceeded 70%, which was far superior to Comparative Example 1 (original sequence). Figure 4 ).

[0066] Kill kinetics experiment: CAR-T cells loaded with chimeric antigen receptors from Example 2 or Comparative Example 2 were co-cultured with tumor cells. The tumor cell lysis process was continuously monitored using a real-time cell analysis system, and the proportion of tumor cell death under a 1:3 effector-to-target ratio was calculated.

[0067] Killing experiments ( Figure 5 The results showed that CAR-T cells loaded with the chimeric antigen receptor of Example 2 (optimized) significantly reduced the EC50 effector-to-target ratio for various target cells and greatly improved the killing efficacy.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A single-chain antibody targeting CD20 and CD19, characterized in that, The amino acid sequence of the single-chain antibody is shown in SEQ ID NO.

1.

2. A chimeric antigen receptor targeting CD20 and CD19, characterized in that, The chimeric antigen receptor includes the single-chain antibody as described in claim 1.

3. The chimeric antigen receptor according to claim 2, characterized in that, The chimeric antigen receptor comprises, from the N-terminus to the C-terminus, a signal peptide, the single-chain antibody of claim 1, a CD8a hinge region, a CD8a transmembrane domain, a CD137 co-stimulatory receptor, and a CD3Z co-stimulatory receptor, in sequence.

4. The chimeric antigen receptor according to claim 3, characterized in that, The amino acid sequence of the signal peptide is shown in SEQ ID NO.2; The amino acid sequence of the CD8a hinge region is shown in SEQ ID NO.3; The amino acid sequence of the CD8a transmembrane domain is shown in SEQ ID NO.4; The amino acid sequence of the CD137 co-stimulatory receptor is shown in SEQ ID NO.5; The amino acid sequence of the CD3Z co-stimulatory receptor is shown in SEQ ID NO.

6.

5. A nucleic acid, characterized in that, The nucleic acid encodes the single-chain antibody of claim 1 or the chimeric antigen receptor of any one of claims 2-4.

6. A carrier, characterized in that, The vector carries the nucleic acid as described in claim 5.

7. A cell, characterized in that, The cell carries the nucleic acid of claim 5, or contains the vector of claim 6, or expresses the single-chain antibody of claim 1 or the chimeric antigen receptor of any one of claims 2-4.

8. The cell according to claim 7, characterized in that, The cells include T cells or macrophages.

9. The use of the chimeric antigen receptor according to any one of claims 2-4 in the preparation of a product for treating lymphoma.

10. The application according to claim 9, characterized in that, The product includes T cells; The T cells express the chimeric antigen receptor as described in any one of claims 2-4.

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