CN114292329B - anti-CD 19 antibodies and uses thereof - Google Patents

Abstract

The invention relates to an anti-CD 19 nanobody, which comprises 3 complementarity determining regions CDR1-3, wherein the sequences are respectively shown as SEQ ID NO. 1-3, the invention carries out nanobody drug development aiming at B cell malignant tumor, and the CDR sequences of the nanobody are identified and humanized nanobody is constructed by preparing human CD19 protein, immunizing alpaca, utilizing platform technology of phage library for displaying nanometer monoclonal antibody and the like, screening out the nanobody VHH which specifically binds CD 19; while using flow cytometry to identify its ability to bind B cell lymphoma cells. The invention provides a potential nano antibody new drug for clinical treatment of B cell malignant tumor.

Description

anti-CD 19 antibodies and uses thereof

Technical Field

The invention relates to the field of biological medicine. More particularly, it relates to an anti-CD 19 antibody and application of the antibody in preparing B cell malignant tumor detection agent and therapeutic drug.

Background

B-cell malignancy is a common hematological malignancy, and although most patients respond to current first-line treatment, the recurrence rate is high, the prognosis is poor, and the B-cell malignancy is still one of refractory malignant tumors. In recent years, CD19 has received great attention as a molecular target for immunotherapy of B cell malignancies.

CD19 is a normal and malignant B lymphocyte specific surface protein, is hardly expressed on the surface of other cells, participates in the regulation of PI3/AKT pathway, and plays a very important role in proliferation and differentiation of B cells. Because of the specificity of CD19 in B lymphocyte expression and the universality of tumor cell expression, and the fact that the CD19 is not lost in the malignant transformation process of the B cell, and hematopoietic stem cells and pro-B cells do not express the CD19, after treatment is stopped, the B cell can be effectively supplemented, so that the CD19 becomes a potential molecular target for the immunotherapy of the B lymphocyte malignant tumor.

In 1993, a novel natural antibody derived from the family camelidae was discovered. The antibody naturally lacks the light chain and consists only of the heavy chain, which comprises two constant regions (CH 2 and CH 3), one hinge region and one heavy chain variable region (Variable heavy chain domain, VHH, i.e., antigen binding site), the relative molecular mass of which is about 13KDa, which is only 1/10 of conventional antibodies, and the molecular height and diameter are both on the nanometer scale, which is the smallest functional antibody fragment available today, and is therefore also known as nanomumab (Nanobody, nb). Due to the characteristics of high stability (not degraded at 90 ℃), high affinity, homology with human antibody of more than 80%, low toxicity and immunogenicity, etc., the nanometer monoclonal antibody is widely used for research and development of immunodiagnosis kit, imaging research and development, and antibody drug research and development in the fields of tumor, inflammation, infectious disease, nervous system diseases, etc.

At present, some CD19-CART cell therapeutic drugs exist in the united states market, but no cell therapeutic drugs for B cell malignancy are currently marketed in China, so that development of antibodies against CD19 is urgently needed for preparing therapeutic drugs for diseases related to CD19, such as B cell malignancy.

Disclosure of Invention

According to the invention, alpaca source nanometer monoclonal antibodies and VHH (very high-frequency binding antigen) are obtained by immunizing alpaca with antigen, and are used for treating B cell malignant tumors. Based on these studies, the present invention provides a nano-antibody capable of binding CD19, comprising 3 CDRs 1-3 with sequences shown in SEQ ID NO. 1-3.

In a specific embodiment, the nanoparticle further comprises 4 framework regions FR1-4, said FR1-4 being staggered with respect to said CDR 1-3. For example, the FR1-4 sequence may be designed as shown in SEQ ID NO. 4-7 (alpaca source), but the scope of the present invention is not limited thereto. The specific recognition and binding capacity of antibodies is largely determined by the CDR region sequences, which are not significantly affected by FR sequences and can be designed according to species, as is well known in the art. FR region sequences of human, murine or camelid origin can be designed to link the CDRs to provide a nanobody that binds CD19.

The invention also provides application of the nano antibody in detecting cell surface CD19.

The invention also provides application of the polypeptide in preparing a medicament for treating CD19 related diseases such as B cell malignant tumor.

The invention also provides application of the polypeptide in preparing CAT cell therapeutic agent.

The invention also provides a nucleic acid coding sequence of the polypeptide and application of the polypeptide in preparing a gene therapy medicament.

In one embodiment, the nucleic acid coding sequence is a DNA coding sequence or an RNA coding sequence.

The invention carries out nano antibody drug development aiming at B cell malignant tumor, screens out nano antibody VHH which specifically binds CD19 by preparing human CD19 protein, immunizing alpaca, utilizing platform technology of phage library to display nano monoclonal antibody and the like, identifies CDR sequence thereof and constructs humanized nano antibody; while using flow cytometry to identify its ability to bind B cell lymphoma cells. The invention provides a potential nano antibody new drug for clinical treatment of B cell malignant tumor.

Drawings

FIG. 1 shows the detection profile of antiserum titers one week after CD19 immunization of alpaca 3.

FIG. 2 shows the flow titer assay of antisera one week after immunization of CD19 with alpaca for one week against CD19 on CD19 expressing Raji cells, RPMI8226 did not express CD19 and was a negative control cell.

FIG. 3 is an electrophoretogram of PCR products amplified using a CD19-VHH phage antibody library as a template.

FIG. 4 is a panned identification of a CD19-VHH phage antibody library, wherein A is a statistical plot of ELISA detection after panned phage library against CD19 protein; b is the second wheel (2 ^nd ) Third wheel (3) ^rd ) And a fourth wheel (4) ^th ) The phage antibody library after panning was selected from 40, 48 clones, respectively, for phage ELISA detection statistics.

FIG. 5 shows the results of flow-through binding assays of C19NB283 antibodies to 293TT cells transiently expressing CD19 membrane proteins.

FIG. 6 shows the results of flow-through binding assays of antibodies C19NB283 to Raji cells.

Detailed Description

1. Preparation of immunogens

According to protein sequence and gene sequence information of human CD19 on NCBI website, polypeptide sCD19 which can effectively induce alpaca to generate specific antibody against human CD19 protein is analyzed and designed, and His-tag (sCD 19-His) or rabbit Fc (sCD 19-rFc) is connected at the C end for subsequent purification and detection.

2. Alpaca immunity and antiserum acquisition

Alpaca was primed with an emulsified mixture of 250 μg sCD19-rf fc protein and 250 μl of freund's complete adjuvant and the antisera titers were detected by blood sampling after 1 week of immunization at day 14, day 28, day 42 with sCD19-rf fc protein and 250 μl of freund's incomplete adjuvant for 3 times, 3 times and 4 times; after 1 week of immunization 4, 100ml was collected for phage antibody library construction.

Antiserum titers were measured by ELISA, plates were coated with sCD19 protein at a concentration of 0.5 μg/ml, 100 μl of gradient diluted antiserum or purified antibody (control was serum from a pre-immune alpaca) was added to each well, incubated for 1.5h at 37 ℃, washed 2 times, and 1: 10000-diluted horseradish peroxidase-labeled anti-Llamma IgG (H+L) secondary antibody, incubating at 37 ℃ for 1H, washing for 4-6 times, adding 100 μl TMB substrate, incubating at 37 ℃ for 10min, and incubating at 50 μl 0.2M H ₂ SO ₄ The reaction was stopped and OD450 nm was measured. ELISA detection of serum titers was defined as being at OD450The blank was more than 2.1 fold and greater than the highest dilution of 0.2.

The results are shown in FIG. 1 and FIG. 2, and FIG. 1 shows that the antiserum titer after 3-priming is 0.36×10 ⁶ . FIG. 2 shows the flow titer of 4-post-immune serum binding to Raji cell surface CD19 at 4X 10 ³ . Thus, the antigen can induce alpaca to produce high titer antisera specific to Raji cell surface CD19 protein.

VHH phage library construction and panning

Collecting 100ml of peripheral blood of the immunized alpaca, separating by using lymphocyte separation liquid (GE Ficoll-Paque Plus) to obtain PBMC of the alpaca, extracting RNA according to a TRIzol operation manual, reversing the RNA into cDNA by using oligo (dT), cloning the VHH gene of the alpaca into phagemid plasmid by using technologies such as primer amplification, molecular cloning and the like, and transforming TG1 bacteria to obtain the VHH phage library. To further identify whether the CD19-VHH phage library was successfully constructed, the VHH gene of interest of the immunized CD19 alpaca was amplified by PCR, and the band of interest was 500bp, which was expected (FIG. 3), indicating that the CD19-VHH phage antibody library contained the VHH gene. Selecting 33 clones for sequencing, wherein sequencing results show that the sequence diversity is 96.9%; the alignment shows that the difference sequences are mostly in the CDR binding regions. The construction gave a CD19-VHH phage antibody library with a library capacity of 1.27X10 ⁹ The positive rate was 100%, the sequence Diversity (Diversity) was 96.9%, and the effective insertion rate (In frame rate) was 94.2%.

Resuscitating of phage antibody libraries was performed with the aid of M13KO7 helper phage, with VHH-phagemid transformed bacteria, and precipitation with PEG/NaCl. The phage antibody library was enriched four times with CD19-His protein coated with 50. Mu.g/ml. And (3) carrying out ELISA (enzyme-linked immunosorbent assay) combination identification on the enriched phage, eluting, converting, plating, picking monoclonal, sequencing clones with the combination reading value and Blank contrast ratio of more than 5, cloning the clones to an expression vector pVAX1, and carrying out transfection on 293F cells to express and produce the nanometer monoclonal antibody.

The library after panning was tested for binding to CD19 protein. Phage ELISA showed that the binding reads of the CD19-VHH phage library to CD19 protein before enrichment was 0.12, and that after one, two and three rounds of enrichment the phage library reads were 0.15, 0.19 and 0.35, respectively (FIG. 4A). To further verify the positive phage rate of binding to CD19-VHH protein in the enriched library, 40, 48 clones were selected from the enriched library of round 2, round 3, round 4, respectively, for single phage ELISA detection. The results showed that in round 2 libraries, 5% of the individual phage clones were positive, 10.4% of the phage clones in round 3 libraries were positive, 39.6% of the phage clones in round 4 libraries were positive, and that the average read of binding was around 3.0 (FIG. 4B), and that the highly bound CD19-VHH phage library was successfully enriched by CD19 protein panning.

Construction of a VHH prokaryotic expression library and VHH expression

Performing PCR amplification on the 4th-CD19-VHH phage antibody library after the four rounds of panning enrichment; obtaining and purifying all the gene fragments of the VHH in the antibody library, cloning the gene fragments of the VHH to a prokaryotic expression vector, converting an SS320 strain, and constructing a prokaryotic expression antibody library of the VHH; the prokaryotic expression antibody library is coated on a flat plate, cultured overnight, 2000 monoclonal colonies are randomly selected the next day, the antibody supernatant is induced to express by using IPTG, and ELISA binding detection is carried out on the antibody supernatant and CD19 protein.

Bacterial supernatants were bound to CD19 protein while no control was bound, and sCD19 bound reads/control reads were greater than 3.0. Wherein the antibody numbered C19NB283 was obtained with a better binding activity to CD19 antibody with an OD450target/OD450 blank value of 32.80. Sequencing shows that the sequences of CDR1-3 of the antibody are shown as SEQ ID NO 1-3, and the sequences of FR1-4 are shown as SEQ ID NO 4-7

Fortebio detection of affinity of VHH antibodies to CD19 protein

Antibody C19NB283 was loaded with an AHC biosensor to detect the affinity level of C19NB283 with CD19 protein. AHC biosensor was equilibrated with 0.02% pbst, loading antibody supernatant, time 200s, again equilibrated with 0.02% pbst, bound CD19 protein, time 180s, dissociated in 0.02% pbst, time 180s, version 8.0 of Fortebio data analysis software, 1:1 binding pattern fitting analysis gave affinity data for C19NB283 with CD19 protein as shown in table 1.

TABLE 1 affinity data for antibodies C19NB283 with CD19 protein

Loading Sample ID

Response

KD(M)

kon(1/Ms)

kdis(1/s)

RMax

Full R^2

C19283

0.1121

1.35E-09

2·04E+05

2.75E-04

0.1164

0·9825

6. Flow cytometry detection of VHH antibody binding to tumor cells

Respectively mixing and incubating C19NB283 with 293TT cells transiently expressing CD19 membrane protein or negative control cells, namely untransfected 293TT cells, 100 μl/sample, and 4 ℃ for 1h; washing with 0.5% PBSF twice, adding fluorescent secondary antibody, and standing at 4deg.C for 30min; after washing twice with 0.5% pbsf, the test was performed on the machine. Flow results showed that the C19NB283 antibody can bind cell surface CD19 protein (fig. 5). Flow assays were performed using Raji cells expressing CD19, with the supernatant that did not express the antibody as a primary negative control, and as a result, C19NB283 was seen to bind to Raji cells (fig. 6). Similar results were obtained using humanized VHH antibodies. Therefore, the antibody C19NB283 has the capacity of targeting and combining B cell malignant tumor, and simultaneously can promote phagocytosis of macrophages by targeting CD19 molecules on the surface of tumor cells, thereby achieving the effect of treating or inhibiting tumor growth, so that the antibody C19NB283 has potential to become a novel antibody medicament for treating tumor.

Because C19NB283 recognizes CD19 molecules on the cell surface, the C19NB283 antibody sequences may also be used in the treatment of CAR (Chimeric Antigen Receptor, antigen chimeric receptor, consisting of VHH sequence fused to third or fourth generation CD28-4-1BB-CD3zeta molecular sequences) cells for the treatment of tumors. In addition, because the C19NB283 can recognize CD19 molecules on the surface of tumor cells, VHH can be used for ADC (Antibody-drug conjugate) treatment through conjugated drugs or conjugated isotopes for molecular image diagnosis depending on antibodies and the like, thereby providing potential nano new drugs for clinical treatment of tumors.

7. In vivo experiments using AAV viral vector loaded humanized VHHs

Adeno-associated virus (AAV) is derived from non-pathogenic wild adeno-associated virus, and is widely used in gene therapy and vaccine research worldwide due to its characteristics of good safety, wide host cell range (dividing and non-dividing cells), low immunogenicity, long time for expressing foreign genes in vivo, etc., which is regarded as one of the most promising gene transfer vectors.

AAV Helper-Free viral packaging systems are available from Cell Biolabs, san Diego USA. Inserting the DNA coding sequence of the VHH into pAAV-MCS plasmid by molecular cloning technology; after successful construction by sequencing, AAV-293T cells were co-transfected with the constructed plasmid pAAV-Ab and pHelper and pAAV-DJ plasmids using PEI transfection reagents in a mass ratio of 1:1:1. Supernatants were collected at 48, 72, 96 and 120 hours after transfection, concentrated with 5xPEG8000 (sigma) and purified with 1.37g/ml cesium chloride. Purified AAV was dissolved in PBS, and the AAV was identified and stored at-80℃after packaging.

ImmunodefimentNOD.Cg-PrkdcsccidIl 2rgtm1Wjl/SzJ (NCG) mice were purchased from Nanjing university model animals, which, like NSG mice, deleted the IL2 receptor gene on the basis of SCID mice, resulting in no mouse T cells, B cells and very few NK cells in vivo. 1.0-15x10 ⁷ PBMC were intraperitoneally injected into NCG mice for 4-6 weeks; three weeks later, blood flow was taken to detect human T cells by staining human CD45 ⁺ 、CD3 ⁺ 、CD4 ⁺ And CD8 ⁺ . The proportion of human CD45 positive cells reached 5% or more, and it was judged that the humanization of the mice was successful. Injecting the mice into the abdominal cavity with Raji cells 3 x10 ⁶ After one week, mice were subjected to AAV-VVH (1X 10 ¹¹ gc/100 μl) intramuscular injection, AAV-GFP was used as a control. The results show that AAV-VVH has therapeutic effects on B cell lymphomas.

The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

Sequence listing

<110> university of Nanjing

<120> anti-CD 19 antibody and use thereof

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 1

Gly Arg Thr Ile Ser Arg Tyr Ala

1 5

<210> 2

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 2

Ile Ser Trp Ser Gly Gly Arg Thr

1 5

<210> 3

<211> 15

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 3

Ala Ala Asp Glu Asp Ile Gly Lys Thr Met His Met Leu Asp Tyr

1 5 10 15

<210> 4

<211> 25

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 4

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 5

<211> 17

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 5

Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Ala Arg Glu Phe Val Ala

1 5 10 15

Ala

<210> 6

<211> 38

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 6

Tyr Tyr Ala Asp Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

1 5 10 15

Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 7

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 7

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

1 5 10

Claims (8)

1. An anti-CD 19 nanobody is characterized by comprising 3 complementarity determining regions CDR1-3, and the sequences of the complementarity determining regions CDR1-3 are shown in SEQ ID NOS 1-3, respectively.

2. The nanobody of claim 1, wherein the nanobody is a VHH of alpaca origin or a humanized VHH.

3. Use of the nanobody of claim 1 or 2 for the preparation of a CD19 detector.

4. The use according to claim 3, wherein the CD19 is cell surface CD19.

5. Use of the nanobody of claim 1 or 2 in the preparation of a medicament for the treatment of B-cell malignancies.

6. Use of the nanobody of claim 1 or 2 in the preparation of a CAR T cell therapeutic.

7. A nucleic acid encoding the nanobody of claim 1 or 2.

8. Use of the nucleic acid of claim 7 for the preparation of a gene therapy drug for a B cell malignancy.