CN106084048A - AntiCD3 McAb single domain antibody - Google Patents

Abstract

The present invention provides an anti-CD3 single domain antibody, the amino acid sequence of which is selected from SEQ ID NO.1 and SEQ ID NO.3. The present invention further provides a nucleotide sequence encoding the anti-CD3 single domain antibody, an expression vector and a host cell comprising the nucleotide sequence. The anti-CD3 single domain antibody provided by the present invention antagonizes the N-terminal portion of human CD3ε, and these antibodies can be used, for example, for the determination of CD3, and other functional proteins that require CD3, such as bispecific antibodies.

Description

anti-CD3 single domain antibody

technical field

The present invention relates to anti-CD3 single domain antibody (sdAb) and its amino acid and nucleotide coding sequence. The present invention also relates to expression vectors and host cells comprising the nucleotide coding sequence.

Background technique

Since the advent of monoclonal antibody technology in 1975, antigen-specific antibodies have transformed many aspects of biology and medicine. In addition to being important research tools, the availability of monoclonal antibodies has opened the way for the development of diagnostics and new treatments for human diseases. However, monoclonal antibodies have technical limitations and disadvantages in the range of applications, such as expensive mammalian-based expression systems.

Over the past decades, a series of new technologies and approaches have been used to improve the performance of traditional antibodies, especially in tumor therapy. For example, adding toxic compounds to existing antibodies, including radioactive isotopes are linked to monoclonal antibodies. However, even today, the amount of monoclonal antibodies used clinically is still very small. Another way to improve the efficiency of tumor killing is to use bispecific antibodies to attract immune cells to tumor cells, thereby killing tumor cells. Most commonly, anti-CD3 antibodies are used to direct T cells to tumor cells. By simultaneously binding CD3 and tumor cell surface antigen (TAA), this bispecific antibody can trigger T cell-mediated tumor cell lysis. The CD3 (cluster of differentiation 3) T cell receptor helps activate cytotoxic T cells. It is a complex protein made up of four different chains. In mammals, contains a CD3γ chain, a CD3δ chain and two CD3ε chains.

Many bispecific antibodies use single-chain antibodies. Single-chain antibodies are the complete minimal antigen-binding fragments derived from conventional IgG molecules. Unfortunately, the yield of scFvs expressed in bacteria is very low. Camels and alpacas contain a unique type of antibody that lacks light chains. These so called heavy chain antibodies have a lower molecular weight than conventional antibodies due to the absence of CH1. The variable heavy chain of this type of immunoglobulin is referred to as VHH to distinguish it from the classic heavy chain variable region. Thus, single-domain VHHs are the smallest available intact antigen-binding 15 kDa fragment, known as a Nanobody. Nanobodies have some unique advantages over Fab, Fv, or single-chain antibodies, such as being more stable and easier to express in bacteria.

The anti-CD3 antibody in the prior art has a single form and cannot meet the actual needs, so it is necessary to develop more anti-CD3 antibodies, so as to provide more options for the production and design of functional proteins.

Contents of the invention

One aspect of the present invention relates to an anti-CD3 single domain antibody, the amino acid sequence of which is selected from SEQ ID NO. 1 and SEQ ID NO. 3.

Another aspect of the present invention relates to a nucleotide sequence encoding the anti-CD3 single domain antibody of the present invention. In one embodiment, the nucleotide sequence is shown in SEQ ID NO. 2. In another embodiment, the nucleotide sequence is shown in SEQ ID NO. 4.

Still another aspect of the present invention relates to an expression vector comprising the nucleotide sequence of the present invention. The invention also relates to host cells comprising the expression vector. In one embodiment, the host cell is E. coli.

The present invention also relates to a bispecific antibody comprising any CD3 single domain antibody described in the present invention.

The anti-CD3 single domain antibody provided by the present invention antagonizes the N-terminal portion of human CD3ε, and these antibodies can be used, for example, for the determination of CD3, and other functional proteins that require CD3, such as bispecific antibodies.

Description of drawings

Figure 1 is a skeleton diagram of the sdAb phage display library.

Figure 2 shows the results of serum titer experiments.

Figure 3. Agarose gel electrophoresis of total RNA isolated from lymphocytes. Lane M, DNA marker Marker III; lanes 1-3, total RNA isolated from lymphocytes from the first blood draw; lanes 4-7, total RNA isolated from lymphocytes from the second blood draw; Lanes 8-10, total RNA isolated from lymphocytes from the third blood draw.

Figure 4. Agarose gel electrophoresis of purified _VHH PCR products. Lane M, DNA marker Marker III; Lane 1-8, using 8 pairs of primers to obtain the purified V _H H PCR product from the PBMC of the first blood draw; Lane 9-16, using 8 pairs of primers to obtain the purified V H H PCR product Purified V _H H PCR products obtained from PBMCs of the second blood draw; Lanes 17-24, purified V _H H PCR products obtained from PBMCs of the third blood draw using 8 pairs of primers.

Figure 5. Insertion rates of sdAb phage display libraries. Ninety-six randomly selected library clones were amplified by PCR using M13R (-48) and M13F (-47) primers. Clones with ~1100 bp DNA bands had _VHH inserts.

detailed description

The present invention will now be further explained in conjunction with the following experiments and accompanying drawings. It should be noted that these experimental examples and accompanying drawings should not be construed as limiting the present invention.

1 strategy

The present invention developed an anti-CD3 single-domain antibody through phage display technology, and carried out the following steps: animal immunity and immune response testing, construction of sdAb phage display library, phage display panning, FASEBA screening and FACS verification.

2 materials

Antigen protein: CD3-His protein (MP-5)

Antigen cell line: Jurkat target cells, KPCN control cells

TRIzol® Reagent (Ambion, Cat. No. : 15596-026)

PrimeScript ^TM 1st Strand cDNA Synthesis Kit (Takara, Cat. No. : 6110A)

Sfi I enzyme (NEB, Cat. No. : R0123S)

Host bacteria: E.coli SS320

Ampicillin, 100 mg/ml

Isopropyl-β-D-thiogalactoside (IPTG), 1 M

PBS: 137 mM NaCl, 2.7 mM _KCl , 4.3 mM Na2HPO4, 1.4 mM _KH2PO4 , pH 7.4

ELISA microtiter plate (Corning, Cat. No. : 9018)

Coating buffer: 0.05 M NaHCO ₃ , pH 9.6

Blocking buffer (PBST): PBS buffer, pH 7.4, add 5% skimmed milk powder

Washing buffer: PBS buffer, pH 7.4, , add 0.05% Tween 20

M13KO7 helper phage (NEB, Cat. No. : N0315S)

HRP/anti-M13 monoclonal antibody (GE Healthcare, Cat. No. : 27-9421-01)

Goat anti-llama IgG[HRP] (Novus Biological, Cat. No. : NB7242)

Rabbit anti-camel IgG [HRP] (GenScript)

FACSCalibur (BD Bioscience, San Jose, CA)

Flowjo: Flowjo 7.6.1 Min software

2×YT: 16 g tryptone, 10 g yeast extract, 5 g NaCl dissolved in 1 L ddH ₂ O.

Octet ^RED 96 (ForteBio)

Super Streptavidin (SSA) Dip and Read ^TM Biosensors (ForteBio)

10 mM Glycine-HCl, pH 2.0

3 methods

3.1 Animal immunity and immune response test

3.1.1 Animal immunization

The CD3-His immunogen was mixed with adjuvant or PBS and injected into llamas. Animals were immunized five times over the course of the project. Peripheral blood samples were collected before immunization and after the 4th and 5th immunizations, respectively. Lymphocytes were isolated by gradient separation. Add RNAlater to cells and store at -80°C. Serum was obtained by centrifugation of anticoagulated blood and stored at -80°C.

3.1.2 Immune response test

The immune response to the immobilized immunogen was assessed by ELISA using serum samples. Sera were evaluated before immunization and after the 4th and 5th immunizations. Antigens (CD3-His and CD34-Fc) were diluted in 4 μg/ml coating buffer, respectively. Coat microtiter plates with diluted antigen overnight at 4°C. Subsequently, the microtiter plate was washed 3 times with wash buffer and blocked with blocking buffer for 2 hours at 37°C. The microtiter plate was washed 4 more times with wash buffer. Serially diluted sera were added to the plate and incubated at 37°C for 2 hr. Microtiter plates were then washed 4 times with wash buffer. Add goat anti-llama IgG [HRP] or rabbit anti-camel IgG [HRP] and incubate for 1 hour at 37°C. After washing, TMB substrate was added to the reactant for 10 minutes, and then 1 M HCl was added to terminate the reaction. MK3 spectrometer measures the absorbance of each well at 450 nm.

3.2 Construction of sdAb phage display library

3.2.1 RNA extraction

Extract total RNA from isolated lymphocytes (see 3.1.1) according to the TRIzol reagent manual. Gel electrophoresis and OD260/280 methods were used to characterize and quantify total RNA.

3.2.2 RT-PCR and _VHH amplification

Total RNA was reverse transcribed into cDNA using oligo(dT)20 primer according to the manual of PrimeScript ^™ 1st Strand cDNA Synthesis Kit. Four sense and two antisense specific degenerate primers were designed to amplify the _VHH fragment, introducing two Sfi I restriction sites. _VHH fragments were amplified according to the GenScript standard operating procedure (SOP).

3.2.3 Display library extension

_VHH PCR products were amplified using different primer pairs. The PCR product was digested with Sfil and gel purified. The gel-purified fragment was inserted into GenScript's own phagemid vector (Figure 1). Build experimental display libraries to optimize ligation and transformation conditions. Optimized linkage and transformation conditions were used to develop the actual display library. A small fraction of transformed cells was diluted and streaked onto 2xYT plates with 100 μg/ml ampicillin. Colonies were counted to calculate library size. Positive clones were randomly picked and sequenced to assess library quality. The remaining transformed cells were streaked onto Ø 15 cm 2 ×YT plates with 100 μg/ml ampicillin and 2% glucose. Scrape the lawn from the plate. A small fraction of cells was used for library plasmid isolation. Glycerol was added to the rest and stored at -80°C for later use.

3.3 Phage library panning

3.3.1 Biopanning

The constructed sdAb phage library was panned for CD3-His protein using the standard procedure developed by GenScript. A large ^sdAb phage display library containing 7.4 x 108 clones (CFU) was used for this screen. Libraries were grown in log phase, recovered with M13KO7 helper phage, and amplified overnight at 30°C on a shaker in 2×YT plates with 100 μg/ml ampicillin and 50 μg/ml kanamycin. Phage were precipitated with PEG/NaCl, resuspended in PBS, and stored at -80°C. For phage panning, microplates (Pierce, Prod# 15100) were coated with CD3-His by incubating them overnight at 100 μg/ml in PBS (pH 7.0) at 4°C. Meanwhile, phage particles were incubated with PBS blocking buffer containing 2% non-fat dry milk for 1 hour at room temperature to block non-specific binding. After rinsing 3 times with PBS, phage particles were added to the microwells and incubated for 1 hour at room temperature on a shaker. After incubation, unbound and non-specifically bound phage were washed away by rinsing the wells 6 times with PBST (0.05% Tween-20 in PBS) followed by 2 times with PBS. The bound phages were immediately used to infect E. coli TG1 cells in logarithmic phase (OD600 about 0.5) for 1 hour at 37°C. After each round of panning, infected cells were mixed with 10% glycerol and then stored at -80°C. For the next round of panning, 10 ml of the infected E. coli TG1 cell stock was added to 30 ml of medium containing 200 μg/ml ampicillin and 2% glucose and grown to logarithmic phase. The culture was aided with M13KO7 for phage recovery, amplified and pelleted phage for the next round of selection. Regular phage display panning was repeated as described above.

3.3.2 Phage ELISA

Individual exported phage clones were grown in 96 deep well plates and screened by phage ELISA to confirm CD3-His specific clones. Coat 96-well plates with 2 μg/ml CD3-His (incubate overnight at 4°C in coating buffer) and block with PBS containing 2% nonfat dry milk. About 50 μl of phage supernatant from overnight cell culture was added per well and incubated for 2 hours at 4°C, followed by four washes with PBST. Unbound sdAb phages were detected by incubation with HRP-labeled anti-M13 mAb for 1 hr at 37°C. Wells were washed an additional four times with PBST and substrate solution was added to each well. Absorbance was measured at 450 nm.

3.4 FASEBA screening and FACS verification

3.4.1 FASEBA screening and affinity grading

The DNA encoding the sdAb fragment of the exported phage was amplified and inserted into the pFASEBA vector to screen for lead antibodies. Individual FASEBA library clones were seeded in 96 deep well plates and induced for expression. ELISA screening was performed to isolate sdAbs that specifically recognized CD3-His protein, and the expression medium for Octet's affinity fractionation was selected. Off-rate screening was performed on an Octet RED96 instrument (ForteBio). All tests were performed at 30°C. The sdAb-SASA protein in the expression medium was captured to the BSA-coated biosensor and incubated with CD3 protein in solution (1x PBS, pH7.4, 0.05% Tween-20). One concentration (100 nM) of CD3 was used for off-rate fractionation. Baseline and dissociation steps were performed in buffer only (1x PBS, pH 7.4, 0.05% Tween-20). Binding kinetics were determined by 1:1 Langmuir binding mode using Fortebio data processing software in kinetic data analysis mode. Binders that interacted with CD3 with high affinity were screened and subjected to DNA sequencing.

3.4.2 FACS verification

To screen for sdAbs that bind Jurkat cells but not KPCN cells, culture supernatants were subjected to flow cytometric analysis. Jurkat cells and KPCN cells were harvested by centrifugation when grown to 70-80% confluency. Seed approximately 4×10 ⁵ cells per well and wash 2 times with PBS. Add 200 µl of culture supernatant to the cells and incubate for 1 h at room temperature. After washing with PBS, antibodies were added to the cells to detect bound sdAb. After the 30 min incubation, wash the cells twice with PBS and resuspend the cells in PBS. Cells were analyzed for sdAb binding using FACSCalibur (BD Bioscience, San Jose, CA) and Flowjo software.

4. Results

4.1 Immune response test

Figure 2 shows the results of serum titer experiments. The titers of the first and second test sera after immunization were much higher than those of the pre-immune sera. No significant difference was observed between the titers of the first and second test sera after immunization.

4.2 Construction of sdAb phage display library

4.2.1 Total RNA extraction

About 449 µg of total RNA was isolated from all harvested lymphocytes (Figure 3), and about half of the total RNA was used to construct a high-quality library.

4.2.2 RT-PCR and _VHH amplification

RT-PCR was performed according to GenScript's SOP using 8 pairs of primers (4 sense primers x 2 antisense primers). Products obtained using different primer pairs were pooled and gel purified (Figure 4). A total of about 24 µg of pure _VHH PCR product was obtained. Common PCR products are used for library construction.

4.2.3 Library Expansion

At least 7.4 x ¹⁰⁷ transformants could be obtained from one set of electrotransformations, so 10 transformations were performed in parallel to obtain the final library. The library size is approximately ~7.4 x 10 ⁸ . According to colony screening, the insertion rate was 97.92% (94/96) (Figure 5). A total of 72 clones were sequenced and 64 clones were in frame. None had the same amino acid sequence (data not shown), indicating the high diversity of the sdAb library.

4.3 Phage library panning

Two rounds of panning and phage ELISA were performed, details of which are listed in Table 1.

Table 1. Details of panning and phage ELISA experiments

Rounds Input (pfu) output (pfu) Phage ELISA positive rate 1 ^st 2.0×10 ¹¹ 3.0×10 ⁵ ~ 5% ^2nd 3.5×10 ¹⁰ 9.7×10 ⁷ ~ 51%

4.4 FASEBA screening and FACS verification

4.4.1 FASEBA screening and affinity grading

DNA encoding the sdAb fragment from the second-round output phage was amplified and inserted into the pFASEBA vector for lead antibody screening by ELISA. 32 clones were obtained by FASEBA ELISA screening. The 32 clones were DNA sequenced and tested for binding to CD3, followed by off-rate analysis by Octet RED96. Combined with the results of FACS (4.4.2), use FortteBio data analysis software to select individual clones in the low dissociation part of the curve. Table 2 shows kinetic data for selected sdAb clones.

Table 2. Kinetic data for sdAb clones

Sample ID serial number k _off (1/s) C57 SEQ ID NO. 1 4.60E-05 C58 SEQ ID NO. 3 3.91E-05

4.4.2 FACS Verification

Jurkat cell binding of supernatants from positive cultures (from 4.4.1) was verified by FACS, and KPCN was used as a negative control cell line. The results of binding affinity grading (4.4.1), binding MFI are listed in Table 3.

Table 3. Binding MFI of culture supernatants to Jurkat cells and KPCN cells*

Sample C57 C58 NC-sdAb TG1 Jurkat cells 213 245 115.03 132.03 KPCN cells 71 84 51 54.5

*: The culture supernatant of NC-sdAb (an unrelated sdAb of the same format) was set as negative control, and the culture supernatant of TG1 was set as background control.

SEQUENCE LISTING

<110> Sun Yat-sen University

<120> Anti-CD3 single domain antibody

<130> 16511CN

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 121

<212> PRT

<213> Artificial sequence

<400> 1

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr

20 25 30

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

35 40 45

Ala Ala Ile Arg Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr

65 70 75 80

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

85 90 95

Ala Ala Ala Gln Lys Ile Asn Thr Asp Val Thr Gly Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 2

<211> 411

<212>DNA

<213> Artificial sequence

<400> 2

caggtgcagc tgcaggagtc tgggggagga ttggtgcagg ctggggactc tctgagactc 60

tcctgtgcag cctctggacg caccttcagt agttatgtca tgggctggtt ccgccaggct 120

ccagggaagg agcgtgagtt tgtagcagct attaggtgga gtggtggtag cacatactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacgatgtat 240

ctgcaaatga acagcctgaa acctgaggac acggccgttt attackgtgc agcagctcaa 300

aagattaata cggatgttac tggtgcctac tggggccagg ggacccaggt caccgtctcc 360

agcggccgct acccgtacga cgttccggac tacggttccg gccgagcata g 411

<210> 3

<211> 130

<212> PRT

<213> Artificial sequence

<400> 3

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr

20 25 30

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

35 40 45

Ser Cys Ile Ser Ser Ser Gly Asp Gly Thr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ala Ala Arg Thr Arg Lys Val Ile Ser Ile Ala Thr Met Thr Leu

100 105 110

Asp Pro Lys Val Phe Ala Ser Trp Gly Gln Gly Thr Gln Val Thr Val

115 120 125

Ser Ser

130

<210> 4

<211> 438

<212>DNA

<213> Artificial sequence

<400> 4

caggtgcagc tgcaggagtc tggaggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag cctctggatt cactttggat tattatgcta tcggctggtt ccgccaggcc 120

ccagggaagg agcgcgaggg ggtctcatgt attagtagcg gtgatggtac cacatactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca atgccaagaa cacggtgtat 240

ctgcaaatga acagcctgaa acctgaggac acggccgttt attackgtgc ggcagcccgc 300

acacgaaaag taatttctat agcgactatg acccttgacc ctaaagtctt tgcttcctgg 360

ggccaggggga cccaggtcac cgtctccagc ggccgctacc cgtacgacgt tccggactac 420

ggttccggcc gagcatag

Claims (8)

1. an AntiCD3 McAb single domain antibody, its aminoacid sequence is selected from SEQ ID NO. 1 and SEQ ID NO. 3.

2. the nucleotide sequence of the AntiCD3 McAb single domain antibody that a kind encodes described in claim 1.

Nucleotide sequence the most according to claim 2, wherein said nucleotide sequence is as shown in SEQ ID NO. 2.

Nucleotide sequence the most according to claim 2, wherein said nucleotide sequence is as shown in SEQ ID NO. 4.

5. an expression vector, it comprises the nucleotide sequence described in any one of claim 2 to 4.

6. the host cell of the expression vector comprised described in claim 5.

Host cell the most according to claim 6, wherein said host cell is escherichia coli.

8. a bi-specific antibody, it comprises the aminoacid sequence of the AntiCD3 McAb single domain antibody described in claim 1.