Patent Application
20090117108
This invention refers to the field of recombinant antibody, more concretely, refers to a recombinant anti-CEA/CD3/CD28 single-chain tri-specific antibody (scTsAb); The method for constructing, expressing and purifying the scTsAb; the vectors and Escherichia coli host cells containing the scTsAb.
The activation of T lymphocytes needs two kinds of signals in vivo: the interaction between MHC/antigen peptide complex on APC (antigen presenting cells) and TCR/CD3 complex on T lymphocytes provides the first signal; the interaction between the co-stimulatory receptor on APC and co-stimulatory molecule on T lymphocytes provides the second signal, that is co-stimulatory signal. It was accepted generally that T lymphocytes cannot be activated fully only in the presence of the first signal (Baxter and Hodgkin, 2002; Bernard et al., 2002)
There are two kinds of T lymphocytes: cytotoxic T lymphocytes (CTL) and T helper cells (TH). CTL is the major effector cell in cellular immunological responses, while TH participate in cellular immunological responses indirectly by secreting cytokines (such interleukin-2 (IL-2)). As tumor immunity majors in cellular immunity, designing anti-tumor drugs to activate CTL specifically is of great importance in tumor immunotherapy. (Foss, 2002)
Now, a series of recombinant anti-tumor/CD3 bispecific antibodies (BsAbs) have been designed to provide the first signal for CTL activation, among which some have entered into clinic research (Daniel et al., 1998; Holliger et al., 1999; Loffler et al., 2000; Manzke et al., 2001a; Manzke et al., 2001b; Dreier et al., 2002; Dreier et al., 2003; Loffler et al., 2003; Min Fang, 2003; Fang et al., 2004). In summary of previous results, BsAbs had been proved to activate T lymphocytes specifically and induce tumor specific cytolysis obviously. However, providing no co-stimulatory signal, most of them could not activate T lymphocytes fully and may result in activation induced cell death (AICD) of T lymphocytes (Daniel et al., 1998), and reduce their tumor specific cytolysis (Daniel et al., 1998).
To overcome above defects, another kind of BsAb was designed: anti-tumor/CD28 BsAb. In company with anti-tumor/CD3 BsAb, they provides CTLs with dual activating signals and induces more efficient tumor specific cytolysis (Jung et al., 2001; Kodama et al., 2002). However, there are several disadvantages for combinatorial using of above two BsAbs, such as the duplicate steps in expression and purification, the consequential increase of production cost, and the partnership of two BsAb in clinical medication. Tri-specific antibody (TsAb) with three binding specificities (to TAA, CD3 and CD28) may replace above two BsAbs in providing dual activating signals in a single molecule and be superior to them in expression, purification and clinical medication.
Heretofore, there are three types of scTsAb: chemical conjugating TsAb (Jung et al., 1991; Tutt et al., 1991; French, 1998; Wong et al., 2000), recombinant polymeric TsAb (Atwell et al., 1999; Dolezal et al., 2000; Schoonjans et al., 2000a; Schoonjans et al., 2000b; Kortt et al., 2001; Schoonjans et al., 2001; Willems et al., 2003) and the type of recombinant single-chain tri-specific antibody (scTsAb)(Li-ping et al., 2003; Zhang et al., 2003). The third type of TsAb is believed to be superior to others for its simplification in construction, expression and purification. Also, as carcinoembryonic antigen (CEA) is a broad-spectrum TAA (Shi et al., 1983; Ganjei et al., 1988; Horie et al., 1996; Kuo et al., 1996; Feil et al., 1999; Tomita et al., 2000; Kammerer et al., 2003), scTsAb containing anti-CEA antibody may be used in preventing or treating diverse tumors in clinic.
The introduction of anti-CEA antibody in anti-CEA/CD3/CD28 scTsAb in this invention provides a convenience to distinguish tumor cells from normal cells in vivo, and avoid or decrease the non-specific killing by activated T lymphocytes.
In another aspect of this invention, as CEA is widely expressed on many tumor cells, it also provides a broad application for treating or preventing different tumor in future. The invention provides an anti-CEA/CD3/CD28 scTsAb for treating or preventing different tumor.
In another aspect of this invention, it provides a method for constructing scTsAb.
The amino acid sequence (SEQ ID NO: 1) of murine anti-CEA single chain fragment of variable region contained in CEA-scTsAb is listed:
The amino acid sequence (SEQ ID NO: 2) of anti-CD3 single chain fragment of variable region contained in CEA-scTsAb is listed:
The nucleic acid sequence (SEQ ID NO: 3) of CEA-scTsAb is listed:
The amino acid sequence (SEQ ID NO: 4) of CEA-scTsAb is listed:
In another aspect of this invention, it provides a vector for expressing CEA-scTsAb: CEA-scTsAb/pTRI.
In another aspect of this invention, it provides an Escherichia coli host cell containing above vector.
In another aspect of this invention, it provides a method for promoting cytoplasmic soluble expression of above scTsAbs at lower temperature.
In another aspect of this invention, it provides a method for purifying above scTsAbs with DEAE anion exchange chromatography.
However, in the context of this invention, other aspects and advantages of this invention are obvious to the ordinary persons engaged in the similar field, especially based on that disclosed in “example” part.
In this invention, all terms are easy to understand for ordinary workers engaged in this field except that is explained specially. Here, some terms are described as below:
Recombinant single-chain tri-specific antibody is the single linear antibody molecule constructed by genetic engineering method with three different antigen binding specificity. To speak concretely, recombinant anti-CEA/CD3/CD28single-chain tri-specific antibody is the single linear molecule constructed by fusing three different antibody fragments (anti-CEA antibody, anti-CD3 antibody, anti-CD28 antibody), interspaced with two inlinkers (FC linker and HSA linker)(Min Fang, 2003). As a alternative, C myc tag and histidine tag can be added at the C terminal of it for activity detection or further purification (Hengen, 1995; Fan et al., 1998) The antibody fragments mentioned here could be single chain fragment of variable region (scFv), Fab fragment of antibody or single domain antibody (VH or VL). More concretely, CEA-scTsAb is constructed by fusing anti-CEA scFv, FC interlinker, anti-CD3 scFv, HSA interlinker and anti-CD28 VH in tandem, with c-myc tag and histidine tag at its C terminal. The advantages for it are listed:
The method for inducing cytoplasmic soluble expression of CEA-scTsAb at low temperature mentioned in this invention, requires that the host bacteria was induced with 0.4 mM IPTG at 30° C. to express CEA-scTsAb solubly in the cytoplasm. With this method, the ratio of inclusion body can be decreased remarkably, and about 50% of expressed CEA-scTsAb is soluble. The soluble expression of CEA-scTsAb can be used in further step of purification directly in no need of denaturation or renaturation, which will make for reducing the cost of production and improving the output.
The method of a single step of purification by collecting flow-through of DEAE anion exchange chromatography, require that the soluble expression product be loaded into the column filled with DEAE anion exchange resin at pH 8.0. Subsequently, almost all none-target proteins can be absorbed, while most of CEA-scTsAb flows through with about 75% purity.
The operating procedure of this invention is listed:
At first, the parent vector pTRI is constructed by introducing a new special Multi-Cloning Sites (MCS). Then the DNA fragment coding anti-CD28 VH is amplified with PCR from the vector, CD28 VH/pTMF, at both ends of which the special pair of restriction sites, Nde I/Kpn I, is added. With the same method, the DNA fragment coding anti-CD3 scFv with the restriction sites, ScaI/SalI, is prepared. The DNA fragment coding anti-CEA scFv with the restriction sites, XhoI/EcoRI, is cut from CEA scFv/pTMF. At last, all above three fragments are introduced into pTRI in tandem to produce the final vector CEA scTsAb/pTRI, in which the DNA fragments coding anti-CEA scFv, anti-CD3 scFv or anti-CD28 VH are arrayed in tandem from N end to C end.
Being transformed into E. coli BL21(DE3) and induced with IPTG at lower temperature (30° C.), CEA-scTsAb is expressed in cytoplasm solubly. With a further single step of DEAE anion exchange chromatography, it is purified primarily. The binding specificities to three antigens (CEA, CD3, CD28) are detected by ELISA; The binding specificities to tumor cells are detected by single color FACS after conjugating CEA-scTsAb with FITC; The cytolysis of tumor cells, the proliferation of T lymphocytes induced by CEA-scTsAb are both analyzed by MTT assay; The morphological changes of tumor cells are recorded by microphotography with inverted microscope. With dual-color FACS, PI/annexin-V-FITC, and fluorescence-microscope, the necrosis and apoptosis of tumor cells induced by CEA-scTsAb are visualized.
This invention will be described in detail below referring to appended drawings. To be comprehended, all examples below are listed here to illuminate the invention, not to restrict it.
To prepare the DNA fragment containing multiple cloning sites by overlapping PCR.
The schematic process is shown in
The operating steps:
Step 1: according to
Reaction mixture: the synthetic fragments, 1 μl (each); 10×PCR buffer, 2 μl; dNTPs (2 mmol/ml each) (Dalian TaKaRa Biotechnology Co. Ltd.), 2111; Taq (1U) (Dalian TaKaRa Biotechnology Co. Ltd.) 0.5 μl; distilled water, 14 μl.
Reaction condition: to pre-denature at 94° C. for 1 minute; to denature at 94° C. for 30 seconds; to anneal at 45° C. for 30 seconds; to elongate at 72° C. for 30 seconds; 10 cycles.
Step 2: according to
To pre-denature at 94° C. for 1 minute; to denature at 94° C. for 30 seconds; to anneal at 45° C. for 30 seconds; to elongate at 72° C. for 30 seconds; 10 cycles.
Step 3: according to
Reaction mixture: the products of step 2, 1 μl (each); 10× PCR buffer, 2 μl; dNTPs (2 mmol/ml each) (Dalian TaKaRa Biotechnology Co. Ltd.), 2 μl; Taq (1U) (Dalian TaKaRa Biotechnology Co. Ltd.) 0.5 μl; distilled water, 13 μl.
Reaction condition: to pre-denature at 94° C. for 1 minute; to denature at 94° C. for 30 seconds; to elongate at 72° C. for 50 seconds; 25 cycles.
Step 4: according to
Reaction mixture: the products of step 3 (UP, DOWN), 1 μl (each); primers (synthetic fragment 1 and 12), 1 μl; 10×PCR buffer, 2 μl; dNTPs (2 mmol/ml each) (Dalian TaKaRa Biotechnology Co. Ltd.), 2 μl; Taq (1U) (Dalian TaKaRa Biotechnology Co. Ltd.) 0.5 μl; distilled water, 12 μl.
Reaction condition: to pre-denature at 94° C. for 1 minute; to denature at 94° C. for 30 seconds; to elongate at 72° C. for 50 seconds; 25 cycles.
The ultimate products (439 bp) are applied to agarose electrophoresis (1%) (
The diagram process of construction is shown in
(1) Construction of pTRI Vector
The DNA fragment containing multiple cloning sites and empty vector pTMF (Zhang et al., 2003) are both cut with NcoI/BarnHI and ligated together. The products of ligating are transformed into E. coli strain TOP10 (Invitrogen). The plasmid isolated from the transformed bacterial cells is named pTRI and used for next step.
Restriction enzyme digesting, ligating, preparation of TOP10 competent cells and transformation are carried out as below:
Restriction enzyme digesting reaction: in a volume of 20 μl, 1 μg of pTMF or the DNA fragment containing multiple cloning sites are digested with NcoI/BamHI (Promega) according to the operating manual. The products are applied to agarose electrophoresis (1%) and purified by DNA Gel purifying Kit (Watson Biotech. Inc.). The digested product for pTMF is about 5000 bp, while that of the DNA fragment containing multiple cloning sites if about 430 bp. Ligating reaction: 50-100 ng cut vector and 3-10 times (mol ratio) cut DNA fragments are mixed in a volume of 20 μl which contained 2 μl 10× T4 DNA ligase buffer, 1U T4 DNA ligase (Dalian TaKaRa Biotechnology Co. Ltd.) and necessary distilled water. The Ligating reaction is carried out at 16° C. overnight.
Preparation of Top10 Competent Cells: to Inoculate the Top10 Bacteria (Invitrogen Co.) to 2 ml LB medium ((10 g/l tryptone (GIBCO Co.), 5 g/l yeast extract (GIBCO Co.), 5 g/l NaCl, pH 7.5)), and incubate overnight at 37° C. with shaking. Then transfer to 20-40 ml LB medium at the rate of 1:100, incubate at 37° C. with shaking to reach A600 0.3-0.4 (about 2.5 hour). To chill on ice for 15 minutes and centrifuge at 4° C. at 4000 rpm for 10 minutes. The pellet is suspended in 10 ml of pre-chilled 0.1 mol/l CaCl2 (Sigma Co.) and chilled on ice for 20 minutes. After the second centrifuge at 4° C. at 4000 rpm for 10 minutes, the pellet is gently suspended in 1˜2 ml of pre-chilled 0.1 mol/l CaCl2 solution with 12% glycerol, and divided the aliquot of 200 μl in each EP tube, stored at −80° C.
Transformation: the ligating mixture is added into 200 μl competent cells. After being mixed gently and chilled on ice for 30 minutes, it is put in water bathe of 42° C. for 100 seconds, and then chilled on ice for 2 minutes. After adding 0.8 ml LB medium into the mixture, to shake it at 37° C. (<150 rpm) for 45 minutes to recover the cells. At last, the cells are centrifuged at 10,000 rpm for 1 minute, re-suspended in 50˜100 μl LB medium, spread onto the LB-K plate (10 g/l tryptone, 5 g/l yeast extract, 5 g/l NaCl, 15 g/l agar (SIGMA Co.), 50 μg/ml kanamycin (SIGMA Co.), pH 7.5) and incubated at 37° C. overnight.
Selection of positive clones: to pick the single clones on the LB-K plate and transfer them into 2 ml LB-K medium (10 g/l tryptone, 5 g/l yeast extract, 5 g/l NaCl, 15 g/l agar (SIGMA Co.), 50 μg/ml kanamycin (SIGMA Co.), pH 7.5) separately. After shaking at 37° C. overnight, the plasmid contained are isolated with Plasmid Isolating Kit. (Watson Biotechnologies, Inc) according to the standard manual. The positive clones are identified by PCR with above isolated plasmids as the templates.
PCR reaction mixture: 0.1˜1 μl plasmid DNA (about 20-200 ng); 10 μmol upstream primer (T7-up: 5′-TAATACGACTCACTATAGGGGA-3′) (SEQ ID NO:17); 10 μmol down stream primer (T7-down: 5′-GCTAGTTATTGCTCAGCGG-3′) (SEQ ID NO: 18); 2 μl 10× Taq buffer; 2 μl 2 mmol/ml dNTPs; 1U Taq; 12 μl distilled-water. The PCR reaction condition: to pre-denature at 94° C. for 5 minutes; denature at 94° C. for 40 seconds; anneal at 53° C. for 40 seconds; elongate at 72° C. for 40 seconds; 25 cycles. At last 5 μl PCR product are applied to agarose electrophoresis (1%). As shown in
The DNA fragment coding anti-CD28VH is amplified from CD28 VH/pTMF (Ju-long et al., 2002)(Cheng et al., 2002) with P1 (P1: 5′-TCACATATGCA GGTACAGC TACAG-3′) (SEQ ID NO: 19) as the up-stream primer and P2 (P2: 5′-TTCGCTAGCGGAAGATACGGTA CCA-3′) (SEQ ID NO: 20) as the down-stream primer. The restriction sites NdeI/NheI are introduced at 5′ end and 3′ end respectively during the process of PCR.
PCR reaction mixture: 1 μl primers (each); 2 μl dNTP (2 mmol/ml each); 2 μl 10× pfu buffer; 100 ng CD28 VH/pTRI, plasmid; 0.3 μl Pfu (Promega Co.); add distilled water to the volume of 20 μl. PCR reaction condition: to pre-denature at 94° C. for 3 minutes; to denature at 94° C. for 30 seconds; to anneal at 55° C. for 30 seconds; to elongate at 72° C. for 50 seconds; 25 cycles. The PCR products of about 350 bp are purified by agarose electrophoresis (1%) and DNA Gel purification kit (Watson Biotech. Inc.).
The above PCR product and pTRI plasmid are cut with NdeI/NheI (Promega Co.) at the same time according to the product manual. The cutting product (about 350 bp) of PCR product and that (about 5300 bp) of pTRI are ligated together and transformed into TOP10 E. coli strain. The plasmids isolated from the positive clones are named as CD28 VH/pTRI, which are identified by PCR with the product of about 750 bp (As shown in
(3) Construction of CD3 scFv/CD28 VH/pTRI The DNA fragment coding anti-CD3 scFv is amplified from CD3 scFv/pTMF (Liu XF, 1996), with P1 (P1: 5′-AAGAGTACTGAGGTGAAGCTGGTGG-3′) (SEQ ID NO: 21) as the up-stream primer and (P2: 5′-GAAGTCGACAGCGCGCTTCAGTTCCAG-3′) (SEQ ID NO: 22) as the down-stream primer. The restriction sites, ScaI and ScaII, are introduced at 5′ end and 3′ end respectively during the process of PCR.
PCR reaction mixture: 1 μl primers (each); 2 μl dNTP (2 mmol/ml each); 2 μl 10× pfu buffer; 100 ng CD28 VH/pTRI, plasmid; 0.3 μl Pfu (Promega Co.); add distilled water to the volume of 20 μl. PCR reaction condition: to pre-denature at 94° C. for 3 minutes; to denature at 94° C. for 30 seconds; to anneal at 55° C. for 30 seconds; to elongate at 72° C. for 50 seconds; 25 cycles. The PCR products of about 750 bp are purified by agarose electrophoresis (1%) and DNA Gel purification kit (Watson Biotech. Inc.).
The above PCR product and CD28 VH/pTRI plasmid are cut with ScaI/ScaII (Promega Co.) at the same time. The cut PCR product (about 750 bp) and that (about 5700 bp) of CD28 VH/pTRI are ligated together and transformed into TOP 10 E. coli strain. The plasmids isolated from the positive clones are named as CD3 scFv/CD28 VH/pTRI, which are identified by PCR with the product of about 1400 bp (As shown in
Construction of anti-CEA scFv by overlapping PCR:
Anti-CEA scFv is designed by Linking VH (the variable region of heavy chain) and VL (the variable region of light chain) of anti-CEA monoclonal antibody (Koga et al., 1990) with a special polypeptide GGGGSGGGGSGGGGS) (SEQ ID NO: 23). The whole amino acid sequence of anti-CEA scFv is back translated into a DNA sequence according to the E. coli preferred codon table (Nakamura et al., 2000), which is spited into 22 complemental oligo-nucleotides. The 22 oligo-nucleotides listed below are synthesized and assembled into the whole DNA fragment coding anti-CEA scFv by overlapping PCR.
The operating steps:
Step 1: according to
Reaction mixture: the synthetic fragments, 1 μl (each); 10× PCR buffer, 2 μl; dNTPs (2 mmol/ml each) (Dalian TaKaRa Biotechnology Co. Ltd.), 2 μl; Taq (1U) (Dalian TaKaRa Biotechnology Co. Ltd.) 0.5 μl; distilled water, 14 μl.
Reaction condition: to pre-denature at 94° C. for 1 minute; to denature at 94° C. for 30 seconds; to anneal at 45° C. for 30 seconds; to elongate at 72° C. for 30 seconds; 10 cycles.
The products are listed: A (1 with 2), B(3 with 4), C(5 with 6), D(7 with 8), E(9 with 10), F(1 with 12), G(13 with 14), H(15 with 16), I(17 with 18), J(19 with 20), K(21 with 22)
Step 2: according to
Reaction mixture: the products of step 1, 10 μl (each 10 pmol).
Reaction condition: to pre-denature at 94° C. for 1 minute; to denature at 94° C. for 30 seconds; to anneal at 45° C. for 30 seconds; to elongate at 72° C. for 30 seconds; 10 cycles.
All products are applied to agarose electrophoresis (1%) and purified by DNA Gel purifying Kit (Watson Biotech. Inc.). The products are listed: a (A with B), b(C), c(D with E), d(F), e(G with H), f(I), g(J with K). The fragments of “a” and “g” are about 120 bp; the fragments of “c” and “e” are about 170 bp; the fragments of “d” and “f” are about 100 bp.
Step 3: according to
Reaction mixture: the product of step 2, 1 μl (each); primers, 1 μl (each); 10× PCR buffer, 2 μl; dNTPs (2 mmol/ml each) (Dalian TaKaRa Biotechnology Co. Ltd.), 2 μl; Taq (1U) (Dalian TaKaRa Biotechnology Co. Ltd.) 0.5 μl; distilled water, 12 μl.
Reaction condition: to pre-denature at 94° C. for 1 minute; to denature at 94° C. for 30 seconds; to anneal at 45° C. for 30 seconds; to elongate at 72° C. for 30 seconds; 25 cycles.
All products are applied to agarose electrophoresis (1%) and purified by DNA Gel purifying Kit (Watson Biotech. Inc.). The products are listed: I (a with b), II (c with d), III (e), IV (f with g). The product I is about 200 bp; the product II is about 250 bp; the product III is about 140 bp; the product IV is about 230 bp.
Step 4: according to
Reaction mixture: the product of step 3, 1 μl (each); primers, 1 μl (each); 10× PCR buffer, 2 μl; dNTPs (2 mmol/ml each) (Dalian TaKaRa Biotechnology Co. Ltd.), 2 μl; Taq (1U) (Dalian TaKaRa Biotechnology Co. Ltd.) 0.5 μl; distilled water, 12 μl.
Reaction condition: to pre-denature at 94° C. for 1 minute; to denature at 94° C. for 30 seconds; to anneal at 45° C. for 30 seconds; to elongate at 72° C. for 30 seconds; 25 cycles.
All products are applied to agarose electrophoresis (1%) and purified by DNA Gel purifying Kit (Watson Biotech. Inc.). The fragment of UP is about 430 bp; the fragment of DOWN is about 340 bp. The products are listed: UP (I with II), DOWN (III with IV).
Step 5: according to
Reaction mixture: the product of step 4, 1 μl (each); primers, 1 μl (each); 10× PCR buffer, 2 μl; dNTPs (2 mmol/ml each) (Dalian TaKaRa Biotechnology Co. Ltd.), 211; Taq (1U) (Dalian TaKaRa Biotechnology Co. Ltd.) 0.5 μl; distilled water, 12 μl.
Reaction condition: to pre-denature at 94° C. for 1 minute; to denature at 94° C. for 30 seconds; to anneal at 45° C. for 30 seconds; to elongate at 72° C. for 60 seconds; 25 cycles.
The product is applied to agarose electrophoresis (1%) and purified by DNA Gel purifying Kit (Watson Biotech. Inc.). The product WHOLE is about 750 bp.
The above schematic process of above operations is shown in
The above PCR product and pTMF plasmid are cut with XhoI/EcoRI (Promega Co.) at the same time. The cutting product (about 750 bp) of PCR product and that (about 5200 bp) of pTMF are ligated together and transformed into TOP10 E. coli strain. The plasmids isolated from the positive clones are named as CEA scFv/pTMF, which are identified by PCR with the product of about 750 bp. All operating procedures needed here come from step (1).
The CEA scFv/pTMF plasmid and the CD3 scFv/CD28 VH/pTRI plasmid are cut with XhoI/EcoRI (Promega Co.) at the same time. The small cut product (about 750 bp) of the former and large one (about 6000 bp) of the latter are ligated together and transformed into TOP10 E. coli strain. The plasmids isolated from the positive clones are named as CEA scTsAb/pTRI, which are identified by PCR with the product of about 2100 bp (As shown in
(1) Transformation of CEA scTsAb/pTRI into BL21 (DE3)(Novagen) E. coli Strain.
The competent BL21 (DE3) cells are prepared referring to the method in example 2. The plasmid CEA scTsAb/pTRI) is isolated with plasmid isolating kit (Watson Biotech. Inc.) according to the manual. The subsequent procedures of transformation and identification of positive clones are performed according to example 2 too.
The single clone of BL21 (DE3) containing CEA-scTsAb/pTRI is pick up from LB-K plate and inoculated in 5 ml LB-K medium. After being cultured at 37° C. with shaking overnight, the culture is transferred into 250 ml LB-K medium at a ratio of 1/100 to shake at 37° C. to reach A600 0.6. IPTG (Takara Biotech. (Dalian)) is added to the final concentration of about 0.4 mmol/l to induce soluble expression at 30° C. for 4 hours. The bacterial cells are harvested by centrifuging at 12,000 rpm for 10 minutes and then re-suspended in phosphate buffered saline (PBS: 8 g NaCl, 0.2 gKCl, 1.44 g Na2HPO4, 0.24 g KH2PO4, pH7.4, 1 liter) (1/5 volume of culture medium). Thus, cytoplasmic soluble CEA-scTsAb is released into the supernatant produced by centrifuging ultrasonic-lyzed cells. Furthermore, soluble expression and inclusion body expression of CEA-scTsAb are detected by reducing SDS-PAGE and Western-blotting according to “molecular cloning: a laboratory manual” (Translated by Jin Dong-yan and Li Meng-feng, 1996, Science Press in China)(Sambrook and Russell, 2001). The expression of CEA-scTsAb in both supernatant and pellet from sonication are detected by SDS-PAGE and Western blot and photographed with Alpha-Image 2200 Documentation and analysis system (American Alpha Innotech Company). As shown in
250 ml culture medium containing bacterial cells expressing CEA-scTsAb are centrifuged at 12,000 rpm at 4° C. for 10 minutes. The pellet is suspended in 50 ml equilibrium buffer (20 mmol/l NaCl, 20 mmol/1 Tris-HCl, pH 8.0) of DEAE anion exchange chromatography for further sonication. After a second centrifugation at 12,000 rpm at 4° C. for 10 minutes, the supernatant containing solubly expressed CEA-scTsAb is applied in purifying step directly.
20 ml of DEAE anion exchange resin (Amersham Bioscience) is suspended in 100 ml equilibrium buffer and packed into a 16×20 cm column (Shanghai Hua-mei). The column is equilibrated with 5 volume of equilibrium buffer at a velocity of 1 ml/minute. The above supernatant is then loaded at a velocity of 0.25 ml/minute. Purified CEA-scTsAb exists in the flow-through fraction. The column is washed or eluted with 2 volume of eluting buffer (500 mmol/I NaCl, 20 mmol/l Tris-HCl, pH 8.0) at a velocity of 0.25 ml/minute and cleaned with 2 volume of 500 mmol/I NaOH at a velocity of 0.5 ml/minute. During the regenerating step, the column is first washed with 2 volume of 1000 mmol/ml NaCl at a velocity of 0.5 ml/minute, and then equilibrated with 2 volume of equilibrium buffer at a velocity of 1 ml/minute for next cycle of purification. At least 4 volume of 20% ethanol should be used to wash resins in avoiding of contamination before storage of the column.
By reducing SDS-PAGE of above flow through fraction, the result of purification is shown in
The purified sample is then dialyzed against PBS at 4° C. overnight, changing dialyzing buffer every 6 hours. The protein concentration is quantified with Bradford method from “Short protocols in molecular biology: a compendium of methods from Current protocols in molecular biology” (Translated by Yan Zhi-ying and Wang Hai-lin, Revised by Jin Dong-yan, 1999, Science Press in China)(Ausubel, 1999). Then, after supplementing sodium azide (0.05% (W/V), Sigma) as the preservative and trehalose (0.15 mol/l, from Microbiology Institute, Chinese Academy of China) as the stabilizer, the product solution is divided into 1 ml aliquot and stored at −80° C.
Preparation of Jurkat cell membrane antigen: 5×106 Jurkat cells (American type culture collection, ATCC, TIB-152) are harvested by centrifuging at 1000 g for 10 minutes. The cell pellet is suspended in 0.5 ml PBS and lysed by ultra-sonication. The supernatant of ultra-sonication produced by centrifuging at 12,000 rpm for 10 minutes is supplemented with sodium azide (0.05% (W/V), Sigma) and trehalose (0.15 mol/l, from Microbiology Institute, Chinese Academy of China), divided into 100 μl aliquots and stored at −80° C.
As shown in
An indirect FACS method is used to detect the binding to various tumor cells. The sources of these tumor cells are listed below.
As shown in
A direct FACS method is used here to test the binding specificity of CEA-scTsAb to PBMC (From Beijing Blood Bank) and Jurkat cells.
As shown in
In summary of example 6 and example 7, CEA-scTsAb could bind to PBMC, Jurkat, and several CEA expressing tumor cells specially.
In the system of in vitro assay of tumor specific cytolysis, CEA expressing tumor cell line, SW1116, is used as the target cell (T), and lymphocytes from PBMC is used as the effector cell (E). After mixing them together at a certain ratio of E/T, CEA-scTsAb is added, tumor specific cytolysis is induced by incubating at 37° C. for 48 hours. The survival level of tumor cells is then tested with MTT assay to evaluate tumor specific cytolysis.
(1) PBMC are prepared according to example 7.
(2) SW1116 cells are cultured and collected according to example 6.
(3) Diluted in 10% FCS containing L15 medium (Gibco Co), SW1116 cells (1×105/ml) are first plated in 96-wells plate (Nunc) with 100 μl/well. Then effector cells (PBMC) are added at different E/T ratio (1, 5, 10) with 100 μl/well. Concentrated CEA-scTsAbs (5 μg/ml) are supplemented with 50 μl/well to reach a final concentration of 1 μg/ml, which is also diluted in 10% FCS containing LI 5 medium (Gibco Co). The mixture is incubated at 37° C. 5% CO2 incubator for about 48 hours. Quadruplicate wells are set for each concentration. The setting of negative controls: no CEA-scTsAb wells for each E/T ratio; the wells containing effector cells only; the wells containing target cells only; the wells containing no cells.
(4) MTT assay: the medium supernatants are removed by aspirating, and the adherent cells are washed with PBS one time. Add 200 μl MTT solution (MTT: (3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide, 500 μg/ml, Sigma) for each well and incubate at 37° C. for 4 hours. Wash the plate one time with PBS and add 200 μl DMSO (Sigma) for each well. Continue to incubate at 37° C. for 30 minutes. Absorbance of each well is measured at a wavelength of 570 nm with background subtraction at 620 nm.
(5) The percent of tumor specific cytolysis is calculated according to the formula:
The percent of tumor specific cytolysis(%)=[A600(ET)−A600(ETA)]/[A600(ET)−A600(M)]×100%
A600(ET): the absorbance of the negative wells without CEA-scTsAb.
A600(ETA): the absorbance of the sample wells.
A600(M): the absorbance of the negative wells containing no cells.
The effect of E/T ratio on tumor specific cytolysis induced by CEA-scTsAb is shown in
After mixing PBMC (effector cells) with SW1116 cells (target cells) in L15 medium (10% FBS) at an E/T ratio of 5, and adding purified CEA-scTsAb at a concentration of 750 ng/ml, the mixture is incubated at 37° C. for 20-40 h in 5% CO2 incubator. Then morphological changes of tumor cells and PBMC are observed under a 40× object lens with an OLYMPUS IMT-2 inverted microscope, and recorded by photomicrography. As shown in
The proliferation of effector cells (mainly T lymphocytes) detected with MTT assay is used to evaluate the activation of T lymphocytes induced by co-incubated tumor cells and CEA-scTsAb.
(1) PBMC are prepared according to example 6.
(2) SW1116 tumor cells are cultured and collected according to example 6 too.
(3) Adjust the concentration of SW1116 cells to about 106/ml in mitomycin C containing (25 μg/ml Sigma) L15 medium and incubate the cell mixture at 37° C. 5% CO2-incubator for 20 minutes. After washing the tumor cells with PBS three times, residual mitomycin C is removed from culture medium.
(4) Adjust the concentration of SW1116 cells to 105/ml and that of PBMC to 5×105/ml in 10% FCS containing L15 medium. Concentrated CEA-scTsAbs (5 g/ml) are supplemented with 50 μl/well to reach a final concentration of 1 μg/ml, which is also diluted in 10% FCS containing L15 medium (Gibco Co). Plate 100 μl of them in 96-well plate and incubate the cell mixture at 37° C. 5% CO2 incubator for 4 days. Quadruplicate wells are set for each concentration of CEA-scTsAb. Negative controls: no CEA-scTsAb wells for each E/T ratio; the wells containing effector cells only; the wells containing target cells only; the wells containing no cells.
(5) MTT assay: the medium supernatants are removed by aspirating, and the adherent cells are washed with PBS one time. Add 200 μl MTT solution (MTT: (3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide, 500 μg/ml, Sigma) for each well and incubate at 37° C. for 4 hours. Wash the plate one time with PBS and add 200 μl DMSO (Sigma) for each well. Continue to incubate at 37° C. for 30 minutes. Absorbance of each well is measured at a wavelength of 570 nm with background subtraction at 620 nm.
(6) The stimuli index (SI) is calculated according to the formula below:
SI=[A600(ETA)−A600(ET)]
A600(ET): the absorbance of the negative wells without CEA-scTsAb.
A600(ETA): the absorbance of the sample wells.
As shown in
To sum up the results from example 4-9, the function CEA-scTsAb focus on two aspects: (1) retargeting effector cells around tumor cells; (2) stimulating effector cells to kill target cells specially. As summarized in
There are three pathway for activated CTLs to kill tumor cells in vivo: activated CTLs secret performs to make holes on the membrane surface of tumor cells, which are broken up and induced to necrosis; grazymes secreted by activated CTLs can enter tumor cells through above holes and induce apoptosis; acitivated CTLs would be induced to express Fas ligands on its surface, which interact with Fas molecules on tumor cells and induce them to apoptosis. PI/annexin-V-FITC dual-color FACS (fluorescence cytometry) and subsequent fluorescence microphotography are used here to distinguish necrosis from apoptosis of tumor cells in in vitro assay of tumor specific cytolysis.
As shown in
In
| Number | Date | Country | Kind |
|---|---|---|---|
| 200410032158.3 | Apr 2004 | CN | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CN2005/000408 | 3/29/2005 | WO | 00 | 1/8/2009 |
