An anti-human CD19 nanobody and its application

Abstract

The present invention discloses an anti-human CD19 nanobody and its applications. The antibody comprises three complementarity-determining regions: CDR1, CDR2, and CDR3. The amino acid sequence of CDR1 is as shown in SEQ NO.1, the amino acid sequence of CDR2 is as shown in SEQ NO.2, and the amino acid sequence of CDR3 is as shown in SEQ NO.3. The anti-human CD19 nanobody preferably comprises the amino acid sequence as shown in SEQ NO.4. Through alpaca immunization, detection of immune responses, and phage display technology, the present invention finally screens out anti-human CD19 nanobodies with strong affinity, which can be used to prepare therapeutic drugs for B-cell related diseases and can also be used to prepare CAR-T cell therapeutics.

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to an anti-human CD19 nanobody and applications thereof. Background Art

[0002] CD19 is a glycoprotein widely expressed on the surface of B cells and is an important marker for B cell development, proliferation, and activation. Due to its key role in B cell-related diseases such as leukemia and lymphoma, CD19 has become an important therapeutic target. Nanobodies are single-domain antibodies derived from camelids. Their small molecular weight, excellent stability, and ease of engineering make them promising for broad application in targeted therapy and bioimaging.

[0003] CD19 nanoantibodies can not only effectively recognize and bind to CD19-positive cells, but also inhibit the growth of tumor cells, and even show significant anti-tumor effects in some animal models.

[0004] CD19 nanoantibodies are mainly used to target B cell-related diseases such as acute lymphoblastic leukemia (ALL) and diffuse large B cell lymphoma (DLBCL). CD19 nanoantibodies can be used as monotherapy or in combination with other treatments (such as chemotherapy, immune checkpoint inhibitors), or in combination with other antibodies.

[0005] When used as a monotherapy or in combination with other treatments (such as chemotherapy and immune checkpoint inhibitors), its application is currently mainly reflected in the following three aspects:

[0006] 1) CAR-T cell therapy: In CAR-T cell therapy, CD19 is an important target, and nanoantibodies can be used to design new CARs to enhance the anti-tumor activity of T cells.

[0007] 2) Diagnosis and Imaging: CD19 nanoantibodies can be used for tumor biomarkers and imaging, helping to accurately locate CD19-positive tumor cells and assist in clinical diagnosis.

[0008] 3) Autoimmune diseases: Due to the key role of CD19 in B cell function, CD19 nanoantibodies may also act as regulators in some autoimmune diseases (such as systemic lupus erythematosus).

[0009] When used in combination with other antibodies, CD19 nanoantibodies can enhance the immune system's attack on tumor cells. Specific effects include:

[0010] 1) Enhanced B cell targeting: CD19 is a unique antigen on the surface of B cells. Targeting CD19 can effectively eliminate B cells, including tumor B cells.

[0011] 2) Synergistic effect: When used in combination with other antibodies, different mechanisms can be exploited to enhance the overall anti-tumor effect. For example, combining with immune checkpoint inhibitors can overcome immunosuppression in the tumor microenvironment.

[0012] 3) Promote immune response: CD19 nanoantibodies can enhance the immune response of T cells and natural killer cells (NK cells) against B cell tumors.

[0013] With a deeper understanding of the immune system, the application scope of CD19 nanoantibodies is expected to continue to expand. Although CD19 nanoantibodies have shown good efficacy in treating certain diseases, their development and use also need to minimize their potential toxic side effects and pathogenic risks, and closely monitor the body's condition during treatment. Summary of the Invention

[0014] To achieve the above objectives, the present invention provides an anti-human CD19 nanobody, which comprises three complementarity determining regions: CDR1, CDR2, and CDR3; the amino acid sequence of CDR1 is shown in SEQ NO.1, the amino acid sequence of CDR2 is shown in SEQ NO.2, and the amino acid sequence of CDR3 is shown in SEQ NO.3.

[0015] SYASG (SEQ NO.1)

[0016] AISWSGRIVSYADSVKG(SEQ NO.2)

[0017] RSGRGDLNDRRSYAY(SEQ NO.3)

[0018] Preferably, the anti-human CD19 nanobody comprises the amino acid sequence shown in SEQ NO.4.

[0019] AVQLVESGGGLVQAGGSLRLSCTASERTFS SYASG WFRQAPGKEREFVA AIS WSGRIVSYADSVKG RFTISRSSAENTVYLQMNSLKPEDTAVYYCSA RSGRGDLN DRRSYAY WGQGTQVTVSS (SEQ NO.4).

[0020] Furthermore, the present invention provides an isolated polynucleotide comprising a nucleotide sequence encoding the anti-human CD19 nanobody.

[0021] Preferably, the isolated polynucleotide comprises the nucleotide sequence shown in SEQ NO.5.

[0022] GCTGTTCAGTTGGTGGAGTCCGGAGGAGGATTGGTGCAGGCTGGTGGTTCTTTGAGGTTGAGTTGCACAGCCTCCGAAAGGACCTTTTCTTCTTATGCCAGTGGGTGGTTTAGGCAGGCCCCGGAAAGGAAAGGGAGTTTGTGGCCGCTATTTCCTGGAGTGGTAGGATCGTCTCCTACGCCGACAG TGTGAAGGGCAGGTTCACCATTAGCAGGTCCAGTGCTGAGAACACCGTTTACCTGCAGATGAACAGCCTGAAGCCCGAGGACACCGCCGTTTACTACTGCAGTGCCAGGTCCGGCAGGGGAGATTTGAACGACAGAAGGTCCTACGCCTACTGGGGACAGGGCACACAGGTCACAGTGAGTCCC(SEQ NO.5).

[0023] Furthermore, the present invention provides a nucleic acid vector comprising the polynucleotide according to claim 1 .

[0024] Furthermore, the present invention provides an isolated cell comprising the polynucleotide as claimed above.

[0025] Furthermore, the present invention provides the use of the anti-human CD19 nanobody in CD19 protein immunoassay, wherein the immunoassay includes immunohistochemistry, immunoblotting and enzyme-linked immunosorbent assay.

[0026] Furthermore, the present invention provides a CD19 protein immunohistochemistry detection reagent, which contains the anti-human CD19 nanobody.

[0027] In addition, the present invention also provides the use of the anti-human CD19 nanoantibody in the preparation of therapeutic drugs for B cell-related diseases, wherein the B cell-related diseases include but are not limited to acute lymphoblastic leukemia (ALL), diffuse large B cell lymphoma (DLBCL), and systemic lupus erythematosus.

[0028] Furthermore, the present invention provides a drug for treating B cell-related diseases, which comprises the anti-human CD19 nanobody described in the present invention.

[0029] Furthermore, the present invention also provides the use of the anti-human CD19 nanobody in the preparation of a CAR-T cell therapeutic agent.

[0030] The present invention uses alpaca immunization and immune response detection, as well as phage library display technology, to ultimately screen out anti-human CD19 nanoantibodies with strong affinity, which can be used to prepare drugs for treating B cell-related diseases and can also be used to prepare CAR-T cell therapeutic agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a statistical graph of alpaca PBMC-H&L (light and heavy chains) titer;

[0032] Figure 2 is the statistical graph of alpaca PBMC-VHH titer;

[0033] Figure 3 It is Alpaca M137-4 th The results of long spots were determined by storage capacity;

[0034] Figure 4 It is Alpaca M137-5 th The results of long spots were determined by storage capacity;

[0035] Figure 5 These are the results of three rounds of solid phase panning phage pool ELISA;

[0036] Figure 6 This is the result of SRP affinity kinetics verification. DETAILED DESCRIPTION

[0037] Example 1 Antibody Immunization

[0038] Alpaca immunization and the use of specific antibodies to detect the strength of the alpaca's immune response to the immune antigen (human CD19 recombinant protein).

[0039] 1. Immunization plan

[0040]

[0041]

[0042] 2. Immunization results

[0043] The serum titers of alpacas against Human CD19-Fc after the fourth and fifth immunizations are shown in Table 1. Figure 1 and Figure 2 shown.

[0044] Table 1 Alpaca serum titer

[0045] Detection of antigens and immune serum H&L titer VHH titer M137 negative serum (100) N / A <![CDATA[M137-4 th Serum 8000 100 <![CDATA[M137-5 th Serum 32000 100

[0046] 3. Experimental conclusions:

[0047] The immune titer of alpaca M137 reached more than 16,000, and the serum titer met the requirements for library construction, indicating that the immunization was successful and subsequent phage library construction and antibody screening can be carried out.

[0048] Example 2 Library Construction

[0049] 1. Technical route

[0050] 1) Isolation of PBMCs after immunization of alpacas;

[0051] 2) Extraction and quality control of PBMC RNA;

[0052] 3) RNA is reverse transcribed into cDNA;

[0053] 4) Amplify VHH using nested PCR;

[0054] 5) Enzyme digestion of the VHH and phagemid, ligation of the VHH to the vector, and electroporation to construct a library;

[0055] 6) Library quality evaluation.

[0056] 2. Experimental Results

[0057] 1) VHH reservoir capacity determination

[0058] Alpaca M137-4 th 、M137-5 th The results of the storage capacity determination are as follows Figure 3 and Figure 4 The formula for calculating reservoir capacity is: reservoir capacity = bacterial liquid volume × dilution factor × number of spots × 10 × positive rate, where M137-4 th The insertion rate was 85% and the total library capacity was 4.08×10 9 ;M137-5 th The insertion rate is 88% and the total library capacity is 8.184×10 9 .

[0059] 3. Experimental Conclusion

[0060] The constructed ANb1337-M137-4+5M phage library has a library capacity of 6.8×10 9 Library sequence analysis showed that the correct insertion rate of VHH was greater than 90%, indicating good diversity.

[0061] Example 3 Library Panning

[0062] The ANb1337-M137-4+5M alpaca immune library was used for panning, and Human CD19-His was used as the antigen. Positive clones were selected through panning and screening methods.

[0063] 1. Selection plan

[0064]

[0065] After panning, Output / Input was used to determine whether specific enrichment occurred.

[0066] 2. Panning results

[0067] The results of the three rounds of panning are shown in Table 2 and Figure 5 shown

[0068] Table 2 Output / Input of three rounds of solid phase panning

[0069] ANb1338-M137-3M Input Output Output / Input Phage titer 1SP 2.00E+12 5.10E+05 2.55E-07 1.00E+12 2SP 2.00E+11 1.50E+07 7.50E-05 9.00E+11 3SP 2.00E+10 6.18E+07 3.09E-03 5.00E+12

[0070] The Output / Input of three rounds of solid phase panning showed specific enrichment.

[0071] A total of 616 single clones were selected, and 347 positive clones were obtained, with a positive rate of 56.3%. All positive clones were sent for sequencing, and a total of 16 unique sequences were obtained, of which 10 positive clones were verified as true positives.

[0072] 3. Experimental Conclusion

[0073] Solid phase panning was performed using the ANb1337-M137-4+5M alpaca immune library as the source and human CD19 as the antigen. Output / Input and Phage pool ELISAs showed that specific enrichment occurred as the panning progressed. Positive clones were screened by ELISA and sequenced to obtain the sequence shown in SEQ NO. 4 of the present invention. This sequence was subjected to SRP affinity kinetics validation, and the results were as follows: Figure 6 As shown, KD = 6.25E-08 (M).

[0074] The above describes in detail the preferred embodiments of the present invention. It should be understood that those skilled in the art can make numerous modifications and variations based on the concepts of the present invention without inventive effort. Therefore, any technical solutions that can be derived by those skilled in the art through logical analysis, reasoning, or limited experimentation based on the concepts of the present invention and the prior art should be within the scope of protection defined by the claims.

Claims

1. An anti-human CD19 nanobody, characterized in that: It includes three complementary determining regions: CDR1, CDR2, and CDR3; the amino acid sequence of CDR1 is shown in SEQ NO.1, the amino acid sequence of CDR2 is shown in SEQ NO.2, and the amino acid sequence of CDR3 is shown in SEQ NO.

3.

2. The anti-human CD19 nanobody according to claim 1, characterized in that It includes the amino acid sequence shown in SEQ NO.

4.

3. An isolated polynucleotide, characterized in that The polynucleotide comprises a nucleotide sequence encoding the anti-human CD19 nanobody according to claim 1.

4. The polynucleotide according to claim 3, wherein It includes the nucleotide sequence shown in SEQ NO.

5.

5. A nucleic acid vector, characterized in that Comprising the polynucleotide according to claim 3 or 4.

6. An isolated cell, characterized in that Comprising the polynucleotide according to claim 3 or 4 or the nucleic acid vector according to claim 5.

7. A CD19 protein immunohistochemistry detection reagent, characterized in that: The reagent contains the anti-human CD19 nanobody as claimed in claim 1.

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