CN118791611A - Single domain antibody targeting CD28 protein and its use - Google Patents

Abstract

The invention belongs to the fields of molecular biology, immunology and medicine, and particularly relates to preparation and application of a single-domain antibody of CD28 protein. The invention obtains single domain antibodies of CD28 protein molecules through screening, wherein the single domain antibodies are respectively named as P200, P196, P151, P156, P121, P223, P231, P266, P318, P347, P393 and P456, and the amino acid sequences are shown as SEQ ID NO. 1-SEQ ID NO. 12; the complementarity determining regions of the single domain antibody are CDR1, CDR2 and CDR3 respectively; the CDR1 amino acid sequences are respectively shown as SEQ ID NO 13-SEQ ID NO 15; the CDR2 amino acid sequences are respectively shown as SEQ ID NO 16-SEQ ID NO 19; the CDR3 amino acid sequences are shown as SEQ ID NO. 20-SEQ ID NO. 25. The CD28 protein molecule single domain antibody provides biomolecules with better antigen binding activity and weaker T cell activating effect for immunotherapy and diagnosis, and simultaneously reduces the risk of causing cytokine storm.

Description

Single domain antibody targeting CD28 protein and its use

Technical Field

The invention belongs to the fields of molecular biology, immunology and medicine, and specifically relates to the preparation and application of a single domain antibody of a CD28 protein.

Background Art

CD28 molecules are members of the immunoglobulin superfamily and are expressed on the surface of CD4+T cells and CD8+T cells in the form of homodimers composed of two peptide chains. CD28 molecules are adhesion molecules on the surface of T cells and are also the most important co-stimulatory molecules. They play a co-stimulatory role by non-covalently binding to the ligands B7-1 (CD80) and B7-2 (CD86) on antigen presenting cells (APCs) to form a B7-CD28 complex. CD28 molecules play a key role in many T cell processes, including cytoskeleton remodeling, cytokine production, survival and differentiation; CD28 molecules, as receptors, interact with the natural ligands B7-1 and B7-2 expressed by APCs, mediating the costimulatory signal required for T cell activation. This signal can increase IL-2 secretion to promote activation, survival and delay T cell dysfunction; blocking the interaction of CD28/B7 may help prevent harmful activation in allergic reactions and autoimmune diseases, while enhancing this interaction can promote tumor rejection. Therefore, CD28 and its signaling pathway may prove to be useful targets for the development of new therapeutic approaches (Michel F. CD28-mediated co-stimulation: a quantitative support for TCR signalling. Nat. Rev. Immunol. 2003; 3: 939-951. Wolchok J.D. Targeting T cell co-receptors for cancer therapy. Immunity. 2016; 44: 1069-1078).

Anti-human CD28 monoclonal antibodies interact with CD28 molecules on the B7-CD28 complex, providing effective co-stimulatory signals, stimulating T cell proliferation, and playing an important role in tumor immunity, autoimmune disease treatment, and immune intervention against transplant rejection. In November 2019, Gary Nabel et al. published experimental data on trispecific antibodies that simultaneously target CD3, CD28, and CD38 proteins in Nature Cancer. In the trispecific antibodies reported in this study, CD28 can be used as an auxiliary signal for T cell activation, and can also be paired with CD38 as a dual target. The antibody is in full-length form, which increases the half-life. Because the dosage of multispecific antibodies is much lower than that of ordinary antibodies, it also saves production costs (Wu, L., Seung, E., Xu, L. et al. Trispecific antibodies enhance the therapeutic efficacy of tumor-directed T cells through T cell receptor co-stimulation. Nat Cancer 1, 86–98 (2020).). In the presence of CD28 co-stimulatory signals, the therapeutic effect of trispecific antibodies on tumors is significantly improved. In January 2020, J.C.Waite et al. reported in Science Translational Medicine that bispecific antibodies targeting CD28 and target antigens enhance the function of bispecific antibodies targeting CD3 and target antigens. The study found that the combination of the two bispecific antibodies showed a synergistic enhancement effect in in vitro experiments. In mouse experiments, the combination of the two bispecific antibodies had obvious advantages over the single bispecific antibodies targeting CD3 and target antigens. This method can not only select the same target, but also different targets to achieve dual targeting, and has good application prospects in the future. (J.C.Waite, B.Wang, L.Haber, A.Hermann, E.Ullman, X.Ye, et al.Tumor-targeted CD28 bispecific antibodies enhance the antitumor efficacy of PD-1 immunotherapy. Science Translational Medicine. 2020.12(549)).

In 1993, Hamers' laboratory found that in addition to conventional tetravalent antibodies, camel serum also contained a large number of molecules similar to immunoglobulins G (IgG). These molecules naturally lack the traditional antibody light chain and part of the heavy chain constant region (CH), but still have strong binding ability to antigens, and all camelids can produce such heavy chain IgG molecules, which are called heavy-chain antibodies (HCAbs). Subsequently, researchers also found heavy chain antibodies that naturally lack light chains and part of the heavy chain constant region CH1 in cartilaginous fish (such as sharks). Hamers laboratory also analyzed and identified the structure and sequence of heavy chain antibodies in camel serum, and found that the antigen "binding region" of heavy chain antibodies is only composed of variable region fragments, which is equivalent to the functional equivalent of traditional antibody antigen binding fragment (Fab). Therefore, people call the antigen recognition region fragment of heavy chain antibodies VHH (variable domain of the heavy chain of heavy-chain antibody, VHH). On this basis, single domain antibodies (sdAb) containing only VHH domains were developed. Since VHH crystals are 2.5nm in diameter and 4nm long, they are also called nanobodies (Nb). The researchers screened and successfully expressed specific single domain antibodies using microbial systems through immunization of camelids or bacterial display technology, and found that they have good biochemical properties. Due to their high specificity binding to target antigens, small molecular weight, strong solubility, strong penetration of biological tissues, and easy modification, they have many applications in the diagnosis and detection of biological products, treatment of pathogenic diseases and cancer treatment. Single domain antibodies (sdAb) Single-domain antibodies (sdAbs) are similar to traditional double-chain antibodies in that they can selectively bind to specific antigenic epitopes. The single heavy-chain antibody variable region (VHH) of a single-domain antibody is a single functional domain that can fully bind to an antigen and is only 12-15 kDa. VHH has a simple structure and has the advantages of low immunogenicity and good permeability when binding to an antigen, as well as the ability to contact more hidden targets that cannot be contacted by conventional antibodies during tumor treatment. In addition, because single-domain antibodies have only one chain, there will be no mismatch problem during the fusion of double-chain antibodies, making them very suitable for the design and construction of bispecific or multispecific antibodies.

Summary of the invention

Based on the specific advantages of single-domain antibodies and CD28 molecules, the present invention obtains single-domain antibodies of CD28 protein molecules by screening, providing biological molecules with better antigen binding activity and weaker T cell activation effect for immunotherapy and diagnosis, while also reducing the risk of causing cytokine storm.

In a first aspect, the present invention provides a single domain antibody of a CD28 protein molecule, wherein the single domain antibodies are respectively named P200, P196, P151, P156, P121, P223, P231, P266, P318, P347, P393, and P456, and their amino acid sequences are shown in SEQ ID NO: 1 to SEQ ID NO: 12;

Furthermore, the complementary determining regions of the single-domain antibody are CDR1, CDR2, and CDR3, respectively; the amino acid sequences of CDR1 are respectively as shown in SEQ ID NO:13-SEQ ID NO:15; the amino acid sequences of CDR2 are respectively as shown in SEQ ID NO:16-SEQ ID NO:19; and the amino acid sequences of CDR3 are respectively as shown in SEQ ID NO:20-SEQ ID NO:25.

Furthermore, the single-domain antibody has the function of improving antigen binding activity and/or reducing T cell activation effect.

Furthermore, the single domain antibody preferably is P156, P196 and P200, which has the function of antigen binding activity and/or reducing T cell activation effect.

In a second aspect, the present invention provides a composition comprising a single domain antibody of the CD28 protein molecule described in the first aspect.

Furthermore, the single-domain antibody composition refers to a composition of a single-domain antibody of a CD28 protein molecule connected to other polypeptides.

Furthermore, the connection can be achieved by genetic engineering or cell engineering methods.

Furthermore, the polypeptide is a polypeptide without antigen binding activity.

Furthermore, the polypeptide is preferably the constant region of human immunoglobulin IgG4 (hIgG4-Fc), and its amino acid sequence (P01861) is shown in SEQ ID NO: 26;

Furthermore, the single domain antibody composition includes but is not limited to the following: such as SEQ ID NO:27-SEQ ID NO:38.

In a third aspect, the present invention provides a pharmaceutical composition comprising a single domain antibody against the CD28 protein molecule of the first aspect and/or a combination of single domain antibodies against the CD28 protein molecule of the second aspect and a pharmaceutically acceptable carrier.

In a fourth aspect, the present invention provides a nucleotide encoding a single domain antibody of the CD28 protein molecule disclosed in the present invention and a composition thereof.

In a fifth aspect, the present invention also provides a vector containing nucleotides of a single-domain antibody comprising a CD28 protein molecule and a composition thereof, and a host cell containing the vector or nucleotides encoding a single-domain antibody comprising a CD28 protein molecule and a composition thereof.

In a sixth aspect, the present invention provides a use of a single domain antibody, composition, nucleotide, vector or host cell of a CD28 protein molecule according to aspects one to six of the present invention in the preparation of a biological agent.

Furthermore, the application includes the application in the preparation of biological products such as diagnosis, detection, treatment of pathogenic diseases and cancer treatment.

Furthermore, the biological products include diagnostic reagents, detection reagents, vaccine preparations, polypeptide products, etc.

Furthermore, the biological agent may have the function of increasing antigen binding activity and/or reducing T cell activation effect.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is an expression purity analysis of the anti-CD28 single domain antibody in Example 1 (Lane M: Protein Marker; Lane 1: LL102-P200, 3.5ug, Reducing; Lane 2: LL102-P200, 2ug, Non-Reducing; Lane 3: LL102-P196, 3.5ug, Reducing; Lane 4: LL102-P196, 2ug, Non-Reducing; Lane 5: LL102-P151, 3.5ug, Reducing; Lane 6: LL102-P151, 2ug, Non-Reducing; Lane 7: LL102-P156, 3.5ug, Reducing; Lane 8: LL102-P156, 2ug, Non-Reducing; Lane 9: LL102-P121, 3.5ug, Reducing; Lane 10: LL102-P121, 2ug, Non-Reducing; Lane 11: LL102-P223, 3.5ug, Reducing; Lane 12: LL102-P223, 2ug, Non-Reducing; Lane 13: LL102-P231, 3.5ug, Reducing; Lane14:LL102-P231,2ug,Non-Reducing; Lane 15:LL102-P266,3.5ug,Reducing; Lane16:LL102-P266,2ug,Non-Reducing; Lane 17:LL102-P318,3.5ug,Reducing; Lane18:LL102-P318,2ug,Non-Reducing; Lane 19:LL102-P347,3.5ug,Reducing; Lane20:LL102-P347,2ug,Non-Reducing; Lane 21:LL102-P393,3.5ug,Reducing; Lane22: LL102-P393, 2ug, Non-Reducing; Lane 23: LL102-P456, 3.5ug, Reducing; Lane24: LL102-P456, 2ug, Non-Reducing).

FIG. 2 is a graph showing the affinity test between the anti-CD28 single domain antibody and the target protein in Example 3

FIG. 3 is a graph showing the affinity test of the anti-CD28 single domain antibody and target cells in Example 3

FIG. 4 is a biological function test of the anti-CD28 single domain antibody in Example 4

DETAILED DESCRIPTION

The term "variable light chain" (VL) as used herein refers to the variable binding region of the antibody light chain. The variable binding region consists of discrete, well-defined subregions, which are called "complementarity determining regions" (CDRs, also known as HVRs (hypervariable regions)) and "framework regions" (FRs). CDRs refer to the amino acids within the antibody variable region that confer antigen specificity and/or binding affinity, separated by FRs. There are three CDRs in each antibody light chain variable region (LCDR1, LCDR2, LCDR3) and three CDRs in each antibody heavy chain variable region (HCDR1, HCR2, HCDR3).

The terms "polypeptide", "peptide" and "protein" used interchangeably herein refer to a polymeric form of amino acids of any length, which may include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. The term includes fusion proteins, including but not limited to fusion proteins with heterologous amino acid sequences, fusions with heterologous and homologous leader sequences, and the like.

As described herein, the term "vector" can also be a nucleic acid molecule capable of transporting another nucleic acid. The vector can be, for example, a plasmid, a cosmid, a virus, a phage, an RNA vector or a linear or circular DNA or RNA molecule, which can include chromosomes, non-chromosomes, semi-synthetic or synthetic nucleic acid molecules. In some embodiments, the vector is a "plasmid", that is, a circular double-stranded DNA loop that can be connected to other DNA segments. In addition, some vectors can guide the expression of the gene to which it is operably connected. Such vectors are referred to as "recombinant expression vectors" (or simply "expression vectors") in this article.

As used herein, the term "recombinant host cell" (or simply "host cell") means a cell into which exogenous nucleic acid and/or a recombinant vector has been introduced. It should be understood that "recombinant host cell" and "host cell" refer not only to the specific subject cell, but also to the progeny of such cells. Transformed host cells may include, but are not limited to, prokaryotic cells, eukaryotic cells, and cells of mammalian, plant, insect, fungal or bacterial origin.

"Isolated nucleic acid" refers to a nucleic acid molecule of a single domain antibody of a CD28 protein molecule in the present invention. Therefore, the term includes, for example, recombinant DNA incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryotic or eukaryotic organism; or as a separate molecule independent of other sequences (e.g., cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion). In addition, the term includes RNA molecules transcribed from DNA molecules, and recombinant DNA that is part of a hybrid gene encoding an additional polypeptide sequence.

The term "variable light chain" (VL) as used herein refers to the variable binding region of the antibody light chain. The variable binding region consists of discrete, well-defined subregions, which are called "complementarity determining regions" (CDRs, also known as HVRs (hypervariable regions)) and "framework regions" (FRs). CDRs refer to the amino acids within the antibody variable region that confer antigen specificity and/or binding affinity, separated by FRs. There are three CDRs in each antibody light chain variable region (LCDR1, LCDR2, LCDR3) and three CDRs in each antibody heavy chain variable region (HCDR1, HCR2, HCDR3).

As used herein, the term "CL" refers to an "immunoglobulin light chain constant region" or "light chain constant region", ie, a constant region from an antibody light chain.

As used herein, the term "expression" refers to the process of producing a polypeptide based on a nucleic acid molecule such as a coding sequence of a gene. The process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof.

Example 1 Single domain antibody screening

1.1 Alpaca Immunity

After cryopreserved human peripheral blood mononuclear cells (PBMCs) (purchased from Miaotong Biotechnology) were revived, the concentration was adjusted to 2×10 ⁶ /ml. Micromagnetic beads loaded with CD3 antibodies and CD28 antibodies (Thermo Fisher Scientific) were added according to the recommended ratio in the instruction manual to activate T cells in PBMCs. After activating T cells, magnetic beads were demagnetized using a magnetic stand; cells were then collected by centrifugation at 1600 rpm for 5 minutes; and cells were cryopreserved using cryopreservation medium. The cryopreservation medium was: RPMI-1640: FBS: DMSO = 4:5:1. The cryopreserved cells were temporarily stored in liquid nitrogen.

Select an adult healthy alpaca. First, thaw the frozen T cells, then wash the cells once with 1*PBS. Resuspend the cells with buffer and immunize the alpaca subcutaneously. Then use the same method to immunize the alpaca five times.

1.2 Phage library construction

After the immunization, 50 mL of peripheral blood was collected from the alpaca after the fourth and fifth immunizations, and the two were mixed to separate peripheral blood mononuclear cells (PBMCs).

Total RNA was further extracted using RNAiso Plus reagent (Takara, catalog number: 9109). The extracted total RNA was reverse transcribed using PrimeScript ^™ II 1st Strand cDNA Synthesis Kit (Takara, catalog number: 6210A) according to the instructions provided by the kit.

Nested PCR was used to amplify the DNA fragment of the variable region of the alpaca heavy chain. Two rounds of nested PCR were performed.

The vector (pComb3XSS) and the target fragment amplified by PCR were digested with SfiI, digested at 50°C overnight, and then the target fragment was recovered. The PCR product after digestion was ligated with the vector, and the molar ratio of ligation was vector:PCR product = 1:3.

The vector connected with VHH was introduced into competent cells by electroporation to construct a heavy chain single domain antibody phage display library. After electroporation, the cells were plated. To detect the insertion rate of the library, 48 clones were randomly selected on the plate for colony PCR.

The results showed that the insertion rate reached 100%. The library capacity was calculated to be 3.92×10 ⁹ by gradient dilution and plating. The bacterial library was inoculated into 2×300mL 2YT+A+G (Amp: 100μg/ml, Glu: 1%) medium until its initial OD600=0.1-0.2, and cultured at 37°C and 230rpm until OD600=0.8 or above. Helper phage M13KO7 was added according to the OD600 value (helper phage: bacteria=20:1) to prepare the phage library. The phage library titer was measured to be 6.56×10 ¹³ cfu/mL by gradient dilution and plating, and the clone number was calculated.

1.3 Screening

The constructed alpaca immune library was affinity screened by solid phase screening to obtain a specific phage library.

1.3.1 Affinity panning

1.3.1.1 Selection

1) Dilute CD28 protein antigen (ACRObiosystems) with carbonate buffer at pH 9.6 to a final concentration of 5 μg/mL. Then add 100 μL/well to the enzyme-labeled wells, coat 8 wells (4 wells for the second round of screening, 2 wells for the third and fourth rounds of screening), and coat overnight at 4°C;

2) Discard the coating solution and wash with PBS for 3 times. Then add 300 μL of 3% OVA-PBS blocking solution to each well and block at 37°C for 1 hour;

3) Discard the blocking solution and wash three times with PBS. Add 100 μL of phage library and incubate at 37°C for 1 hour;

4) Aspirate unbound phages, wash 6 times with PBST and 2 times with PBS;

5) Add 100 μL of Gly-HCl eluent to each well and incubate at 37°C for 8 min to elute the specifically bound phages; 6) Transfer the eluent to a 1.5 mL sterile centrifuge tube and quickly neutralize with 15 μL of Tris-HCl neutralization buffer;

7) Take 10 μL of the neutralized solution for gradient dilution, determine the phage titer, and calculate the panning recovery rate.

8) The remaining eluates were mixed, amplified and purified for the next round of affinity panning.

The panning conditions are shown in Table 1.

Table 1 Affinity panning conditions

1.3.1.2 Library amplification after panning

After panning, the library needs to be amplified. The main steps are as follows:

1) The panning eluate was mixed with 20 mL of E. coli TG1 culture in the early logarithmic growth stage, incubated at 37°C for 30 min, 1 ml of 20% glucose was added, and the culture was shaken at 220 r/min for 30 min. Then, M13K07 phage and 4 μL Amp+ were added at a ratio of cell: phage = 1:20, incubated at 37°C for 30 min, and 20 ml of 2YT liquid culture medium was added, and the culture was shaken at 220 r/min for 30 min;

2) The culture was divided into centrifuge tubes and incubated at 4°C, 5000 rpm for 10 min. The cell pellet was resuspended in 50 mL of 2×YT-AK liquid medium and cultured at 30°C, 250 rpm overnight;

3) Centrifuge the overnight culture at 4°C, 10,000 rpm for 20 min, transfer the supernatant to a new centrifuge tube, add 1/5 volume of PEG-NaCl, mix well, and place at 4°C for more than 2 h;

4) 4°C, 10000 r/min, 20 min, remove the supernatant, resuspend the precipitate in 1 mL PBS, add 1/5 volume of PEG/NaCl, mix well, and place at 4°C for more than 1 h;

5) 4°C, 12000 r/min, 2 min, remove the supernatant, suspend the precipitate in 200 μL PBS, which is the amplification product, measure the titer, and use it for the next round of selection or analysis.

1.3.1.3 Phage rescue

1) From the plate for panning eluate titer, a total of 480 clones were randomly selected from the second, third, and fourth round titer determination plates using a sterilized toothpick, inoculated into 300ul 2×YT-A, and cultured at 37°C, 230r/min, and shaken for 8h.

2) Take 100 μL of the above culture, add M13K07 phage at a ratio of cell: phage = 1:20, incubate at 37°C for 15 min, and shake at 220 rpm for 45 min.

3) Add 300 μL of 2×YT-AK and culture at 30°C with vigorous shaking overnight.

4) The next day, centrifuge at 12000 rpm for 2 minutes.

5) Take the supernatant and use it for monoclonal ELISA identification.

1.3.1.4 Identification of positive phage clones

1) Dilute the target antigen with carbonate buffer at pH 9.6 to a final concentration of 2 μg/mL and

5 μL/well was added to the enzyme-labeled wells and coated overnight at 4°C;

2) Discard the coating solution and wash with PBST three times;

3) Add 300 μL of 5% skim milk to each well and block at 37°C for 1 h;

4) Wash once with PBST, add 50 μL of phage culture supernatant and 50 μL of 5% skim milk to each well, and incubate at 37°C for 1 h;

5) Wash with PBST for 5 times, then add horseradish peroxidase-labeled anti-M13 antibody (diluted 1:10000 in PBS), 100 μL/well, and incubate at 37°C for 1 h;

6) Wash the plate 6 times with PBST. Add TMB colorimetric solution for color development, 100 μL/well, 37°C, 7 min;

7) Add stop solution to terminate the reaction, 50 μL/well, and measure the optical density at 450 nm.

Example 2 Antibody Expression and Purification

2.1 A total of 12 good candidate anti-CD28 single domain antibody sequences were screened by the above method. The present invention named these 12 single domain antibodies as: P200, P196, P151, P156, P121, P223, P231, P266, P318, P347, P393, P456. The variable region amino acid sequences of these single domain antibodies are shown in Table 2.

Table 2 Amino acid sequences and CDR sequences of single domain antibodies

The CDR regions of these single-domain antibodies were analyzed using the IMGT database to obtain the CDR1, CDR2 and CDR3 amino acid sequences of each single-domain antibody, see Table 2.

2.1 Obtain the constant region amino acid sequence of human immunoglobulin IgG4 (hIgG4-Fc) (P01861) from the Uniprot database SEQ ID NO: 26:

ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD KSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

2.3 The 12 CD28 single-domain antibody VHH amino acid sequences were connected to the hIgG4-Fc amino acid sequence respectively to obtain 12 CD28 VHH-Fc fusion proteins, which were named as: LL102-P200, LL102-P196, LL102-P151, LL102-P156, LL102-P121, LL102-P223, LL102-P231, LL102-P266, LL102-P318-Fc, LL102-P347, LL102-P393, and LL102-P456.

2.4 The gene sequence of the anti-CD28 single domain antibody VHH-Fc fusion protein was obtained by gene synthesis. Then, 12 anti-CD28 VHH-Fc fusion protein genes were cloned into the expression vector pCDNA4 (Invitrogen, CatV86220). The anti-CD28 single domain antibody VHH-Fc fusion proteins were expressed respectively by transient transfection of CHO suspension cells.

2.5 After the expression is completed, the supernatant is collected and purified using a Protein A affinity chromatography column to finally obtain the purified CD28 single domain antibody Fc fusion protein.

The amino acid sequences of the 12 candidate anti-CD28 single domain antibody VHH-Fc fusion proteins are: LL102-P200 (SEQ ID NO: 27):

EVQVVESGGSLRLSCPASGRFSRYAMGWFRQAPGKEREFVSAVSRSGGTTSYSDSVKGRFTISRDDNAKNTVYLQMNSLKPEDTAVYYCAIGSSGLVSTREGEYDYWGQGAQVTVSS.

ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

LL102-P196 (SEQ ID NO: 28)

QVQLVESGGGLVQTGGSLRLSCAASGSTSTIRDMDWYRQGPRKQREWVAGVTSGGATGYAPSVKDRFT

ISRDNAKNTLDLQMNSLNIEDTAVYYCTALVAGSGRRWGQGTQVTVSS

ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

LL102-P151 (SEQ ID NO: 29)

EVQLVESGGLVQTGGSLRLSCPASGRFSRYAMGWFRQAPGKEREFVSAVSRSGGTTSYSDSVKGRFTISR

DNAKNTVYLQMNSLKPEDTAVYYCAIGSSGLVSTREGEYDYWGQGAQVTVSS

ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

LL102-P156 (SEQ ID NO: 30)

EVQVVESGGGLVQAGGSLRLSCAASGSTTSTIRDMDWYRQGPRKQREWVAGVTSGGATGYAPSVKDRF

TISRDNAKNTLDLQMNSLNIEDTAVYYCTALVAGSGRRWGQGTQVTVSS

ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK

TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

LL102-P121(SEQ ID NO:31)QVQLVESGGGLVQGGGSLRLSCAASGTSTSSIRDMDWYRQGTRSQREWVAGVSRGGATGYAPSVKDRFTISRDNANNTLYLQMNSLKPEDTAVYYCYVRGFTPAGWKDFWGQGTQVTVSSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

LL102-P223 (SEQ ID NO: 32)

QLQLVESGGGLVQAGGSLRLSCAASGSTSTIRDMDWYRQGPRKQREWVAGVTSGGATGYAPSVKDRFTISRDNAKNTLDLQMNSLNIEDTAVYYCHARTFNSSPYWGKGTLVTVSS

ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD KSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

LL102-P231 (SEQ ID NO: 33)

QLQLVESGGGLVQPGGSLRLSCVASGSTSTIRDMDWYRQGPRKQREWVAGVTSGGATGYAPSVKDRFTISRDNAKNTLDLQMNSLNIEDTAVYYCTALVAGSGRRWGQGTQVTVSA

ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD KSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

LL102-P266 (SEQ ID NO: 34)

QLQLVESGGGLVQSGGSLRLSCAASGTSTSSIRDMDWYRQGTRSQREWVAGVSRGGATGYAPSVKDRFTISRDNAKNTLDLQMNSLKPEDTAVYYCTALVTGSGRRWGQGTQVTVSV

ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD KSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

LL102-P318 (SEQ ID NO: 35)

EVQVVESGGGLVQAGGSLRLSCAASGSTSTRDWYRQGPRKQREWVAGVTSGGATGYAPSVKDRFTISRDNAKNTMDLQMNSLKPEDTAVYYCYARRYTPSGWKDFWGQGTQVTVSSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

LL102-P347 (SEQ ID NO: 36)

QLQLVESGGGLVQGGGSLRLSCAASGSTSSIRDMDWYRQGTRSQREWVAGVSRGGATGYAPSVKDRFTISRDNAKNTLDLQMNSLKPEDTAVYYCNARRGGMNYWGKGTLVTVSS

ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD KSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

LL102-P393 (SEQ ID NO: 37)

EVQLVESGGGLVQPGGSLRLSCAAVGSTSTIRDMDWYRQGPRKQREWVAGVTSGGATGYAPSVKDRFTISRDNAKNTLDLQMNSLNIEDTAVYYCTALVAGSGRRWGQGTQVTVSS

ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD KSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

LL102-P456 (SEQ ID NO: 38)

QLQLVESGGGLVQAGGSLRLSCAASGSTSTIRDMDWYRQGPRKQREWVAGVTSGGSTGYAPSVKDRFTISRDNAKNTLDLQMNSLNIEDTAVYYCTALVAGSGRRWGQGTQVTVSS

ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD KSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

Through patent research, two published anti-CD28 antibody sequences were obtained (reference documents WO2022061098A1 and WO2020210392A1), respectively named: LL102-BMK1 and LL102-BMK2, and the amino terminal sequences are: LL102-BMK1 heavy chain amino acid sequence (SEQ ID NO: 39):

EVQLVESGGGLVQPGGSLRLSCAASGFTFSRNNMHWVRQAPGKGLEYVSGISSNGGRTYY

ADSVKGRFTISRDNSKNTLYLQMGGLRAADMAVYFCTRDDELLSFDYWGQGTLVTVSS

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY

SLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV

ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD

GVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK

GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD

GSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

LL102-BMK1 light chain amino acid sequence (SEQ ID NO:40):

DIQMTQSPSSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLlYAASSLQSGVPSRFS

GSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS

KDSTYSLSSTLLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

LL102-BMK2 heavy chain amino acid sequence (SEQ ID NO:41):

QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWIGSIYPGNVNTNY

AQKFQGRATLTVDTSISTAYMELSRLRSDDTAVYYCTRSHYGLDWNFDVWGKGTTVTVSS

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY

SLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV

ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD

GVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK

GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD

GSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

LL102-BMK2 light chain amino acid sequence (SEQ ID NO:42):

DIQMTQSPSSSLSASVGDRVTITCQASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSR

FSGSGSGTDFTLTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIK

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS

KDSTYSLSSTLLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

2.6 Antibody protein concentration and total protein amount

After the purification of the anti-CD28 single-domain antibody VHH-Fc fusion protein is completed, the protein concentration and total protein amount of different antibodies are detected by ultraviolet spectrophotometry, and then the expression amount of each antibody is calculated according to the expression volume. The absorbance value A280 of the sample solution is read at a wavelength of 280nm using NanoDrop1000, and the protein concentration of the sample is calculated by the formula C (mg/mL) = A280/ε (ε is 1.482mL/mg·cm-1). The purity of different antibodies is evaluated by reducing and non-reducing SDS-PAGE (Polyacrylamide gel electrophoresis) gel electrophoresis. The basic steps are as follows: The samples are electrophoretically separated using Invitrogen's Xcell Surelock Novex Mini Cell electrophoresis tank and Nupage 4-12% Bis-Tris Gel gradient gel. The sample was diluted to about 1 mg/mL, and appropriate amount of reducing agent, loading buffer and pure water were selectively added. After mixing, it was heated at 70°C for about 10 minutes. The loading amount was 2-10 μg, the electrophoresis voltage was about 200 V, and the electrophoresis time was about 35 minutes. After the electrophoresis, Coomassie brilliant blue staining was used for about 2 hours, and the decolorization time was about 3 hours. After decolorization, the Alpha View-Alpha Imager SA software gel imaging system of ProteinSimple was used to take pictures and analyze and calculate the purity of the main band (Figure 1).

The results showed that the anti-CD28 single domain antibody VHH-Fc fusion protein expressed by the present invention had good purity.

2.7 The present invention also uses size exclusion high performance liquid chromatography (SEC-HPLC) to evaluate the purity of different antibodies.

The basic steps are as follows: dilute the sample to about 1.0 mg/mL, use a TSKgel G3000SWXL column, set the column temperature to 25°C, use 100mM phosphate buffer, 100mM sodium sulfate, pH 7.0±0.2 as the mobile phase, the injection volume is 20-50μL, at a flow rate of 1.0mL/min, isocratic elution for 20min, detection at a wavelength of 280nm, and the peak area normalization method is used to obtain the monomer content. The expression amount and purity information of different anti-CD28 single-domain antibody VHH-Fc fusion proteins are shown in Table 3.

Table 3 Expression level and purity of anti-CD28 single domain antibody

The above results show that the anti-CD28 single domain antibody VHH-Fc fusion protein expressed by the present invention has good purity and expression level.

Example 3 Anti-CD28 single domain antibody affinity detection

3.1ELISA experiment

3.1.1 An ELISA experiment was performed according to the conventional experimental process to detect the binding ability of the anti-CD28 single domain antibody of the present invention with the target protein. The specific implementation steps are as follows:

1) hCD28 protein (purchased from Beijing Biopsies, catalog number CD8-H52Hc) was diluted to 1 μg/mL using PBS (purchased from Hyclone) and added to a 96-well ELISA plate at 100 μL/well for antigen coating;

2) Place the cell culture plate in a 37°C constant temperature incubator and incubate for 60 min;

3) After incubation, wash the plate three times with PBST (PBS solution containing 0.05% Tween20), and add PBS containing 2% BSA at 200 μL/well for blocking;

4) Incubate the cell culture plate at room temperature for 60 minutes;

5) Dilute the different groups of LL102-VHH-Fc fusion protein samples of the present invention and the control antibodies LL102-BMK1 and LL102-BMK2 to 1 μg/mL with diluent (PBS containing 2% BSA), and dilute them in a 2.5-fold gradient to 0.04 ng/mL on a sample dilution plate;

6) Add the sample to a 96-well ELISA plate at 100 μL/well and incubate in a 37°C incubator for 60 min;

7) After the incubation, wash the plate three times with PBST, and dilute the secondary goat anti-human IgG (Fc specific)-HRP antibody (purchased from Sigma, catalog number A-0170) 5000 times with diluent;

8) Add 100 μL/well of the ELISA plate and incubate in a 37°C incubator for 30 min;

9) After incubation, wash the plate three times with PBST and aspirate the supernatant;

10) Add 100 μL/well of colorimetric solution, which is 100 μg/mL TMB (3,3',5,5'-tetramethyl-10-benzidine); 11) Incubate in a 37°C constant temperature incubator in the dark for 10 min;

12) Add stop solution (2M/L hydrochloric acid solution) at 100 μL/well and use a microplate reader (Thermo Fisher Scientific, Varioskan LUX) to detect the absorption value at 420 nm/620 nm;

13) Analyze data using GraphpadPrism.

As shown in Figure 2, the EC50 values of LL102-BMK1 and LL102-BMK2 binding to hCD28 protein are 41.4 ng/mL and 91.2 ng/mL. The EC50 values of the antibodies in the present invention binding to the target protein are shown in Table 4. The results show that the binding activity of the antibody molecules of the present invention is significantly better than that of the control antibody LL102-BMK2, and the EC50 value is about 1/4 to 1/2 of that of LL102-BMK2; except for LL102-P151, P223, and P347, the affinity activity of the remaining molecules is better than that of the control antibody LL102-BMK1, and the EC50 values are lower than LL102-BMK1.

In summary, the antibody molecule of the present invention exhibits good CD28 antigen binding activity at the protein level, reaching an affinity level significantly higher than or equivalent to that of the control molecule.

Table 4 Affinity detection of anti-CD28 single domain antibody and target protein

3.2 Affinity detection of anti-CD28 single domain antibody for Jurkat cells

1) Resuspend Jurkat cells in FACS Buffer (PBS containing 2% FBS) and add 10,000 Jurkat cells to a 96-well U-bottom culture plate at 50 μL/well;

2) Dilute the different groups of LL102-VHH-Fc fusion protein samples of the present invention, the positive control antibodies LL102-BMK1, LL102-BMK2, and the negative control antibody IgG1 Isotype to 200 μg/mL with FACS Buffer, and dilute them in 8 gradients in a 3-fold gradient on a sample dilution plate;

3) Take 50 μL of dilution solution and add it to the corresponding well, mix it with the cells evenly, and the final maximum concentration is 100 μg/mL;

4) Place the culture plate in a 4°C refrigerator and incubate for 60 min. After incubation, wash the cells twice with PBS.

5) Dilute the secondary antibody PE anti-human IgG Fc antibody (purchased from BioLegend, catalog number 410708) 100 times with FACS Buffer, add 100 μL/well into the U-bottom plate and mix well with the cells;

6) Incubate at 4°C for 30 min. After incubation, wash the plate twice with PBS and add 100 μL FACS Buffer to resuspend the cells.

7) Use flow cytometry to detect the proportion of PE channel positive cells;

8) Analyze the flow cytometry results using Flowjo and derive the mean fluorescence intensity (MFI) of cell-bound antibodies;

9) GraphpadPrism software was used to fit the dose-MFI value nonlinear curve.

The results are shown in FIG3 , which show that the antibody LL102-P156 of the present invention has strong binding activity with the CD28 protein on the surface of Jurkat cells, and the binding levels of the remaining antibody molecules are comparable to those of the positive controls LL102-BMK1 and LL102-BMK2.

Example 4 Biological function detection of anti-CD28 single domain antibody

1) Dilute human CD3 antibody (purchased from Invitrogen, Catalog No. 2265448) to 1 μg/mL using PBS, and add 100 μL/well to a 96-well white cell culture plate (purchased from Thermo, Catalog No. 136101) for coating;

2) Place the cell culture plate in a 4°C refrigerator and incubate overnight;

3) After incubation, wash the plate three times with PBS;

4) Resuspend the cells with diluent (1640 medium containing 2% FBS), and add 60,000 reporter gene cell line Jurkat-NFAT-Luciferase cells (Smith-Garvin J.E. et al., 2009. T Cell Activation. Ann. Rev. Immunol. 27: 591-619.) into a 96-well culture plate at 50 μL/well;

5) different LL102-VHH-Fc fusion proteins of the present invention, positive control antibodies LL102-BMK1, LL102-BMK2, and negative control antibody IgG1 Isotype were diluted to 200 μg/mL with diluent, and diluted in 3-fold gradients to 8 points on a sample dilution plate;

6) Take 50 μL of the dilution and add it to the corresponding wells of the cell culture plate. The final maximum working concentration is 100 μg/mL;

7) Place the cell culture plate in a 37°C constant temperature incubator and incubate for 4-6 hours;

8) After the incubation, add D-luciferin reaction solution (purchased from Thermo Fisher Scientific, catalog number L2912) into the culture plate at 100 μL/well;

9) After reacting at room temperature in the dark for 10 minutes, the fluorescence signal value (RLU) was detected using an enzyme reader (Thermo Fisher Scientific, Varioskan.LUX); 10) Graphpad Prism software was used for data analysis and fitting of the dose-dependent curve.

The results showed that the antibody molecules of the present invention had weaker activation effects on T cells than the positive control LL102-BMK2. The activation levels of LL102-P151, P156, P196 and P200 molecules on T cells were comparable to those of LL102-BMK1, while LL102-P318, P347, P393 and P456 molecules were significantly stronger than LL102-BMK1 (see Figure 4).

In summary, LL102-P156, P196 and P200 molecules have better antigen binding activity and weaker T cell activation effect, which reduces the risk of causing cytokine storm and is worthy of further evaluation and modification.

Claims (10)

1. A single domain antibody of CD28 protein molecule is named as P200, P196, P151, P156, P121, P223, P231, P266, P318, P347, P393 and P456 respectively, and the amino acid sequence of the single domain antibody is shown as SEQ ID NO. 1-SEQ ID NO. 12.

2. The single domain antibody of claim, wherein the complementarity determining regions of the single domain antibody are CDR1, CDR2, CDR3, respectively; the CDR1 amino acid sequences are respectively shown as SEQ ID NO 13-SEQ ID NO 15; the CDR2 amino acid sequences are respectively shown as SEQ ID NO 16-SEQ ID NO 19; the CDR3 amino acid sequences are shown as SEQ ID NO. 20-SEQ ID NO. 25.

3. A composition comprising a single domain antibody of the CD28 protein molecule of claim 1.

4. A composition of single domain antibodies to CD28 protein molecules according to claim 3, wherein said single domain antibody composition is a composition of single domain antibodies to CD28 protein molecules linked to other polypeptides.

5. A composition of single domain antibodies to CD28 protein molecules according to claim 4, wherein said polypeptide is selected from the group consisting of the constant region of human immunoglobulin IgG4 (hIgG 4-Fc) and the amino acid sequence of said composition is shown in SEQ ID NO. 27-SEQ ID NO. 38.

6. A pharmaceutical composition comprising the composition of claims 1-3 and a pharmaceutically acceptable carrier.

7. A single domain antibody encoding a CD28 protein molecule and the nucleotides of a composition thereof.

8. A vector comprising nucleotides of a single domain antibody of a CD28 protein molecule and compositions thereof, and a host cell comprising the vector or nucleotides encoding a single domain antibody of a CD28 protein molecule and compositions thereof.

9. Use according to the invention as claimed in claims 1 to 5 for the preparation of biological agents.

10. The biological agent of claim 9, which can have a function of increasing antigen binding activity and/or attenuating T cell activating effect.