Construction and selection of the natural immune Fab antibody phage display library from patients with colorectal cancer

Abstract

AIM: To construct the natural immune Fab antibody phage display libraries of colorectal cancer and to select antibodies related with colorectal cancer.

METHODS: Extract total RNA from tissue of local cancer metastasis lymph nodes of patients with colorectal cancer. RT-PCR was used to amplify the heavy chain Fd and light chain κ and the amplification products were inserted successively into the vector pComb3 to construct the human libraries of Fab antibodies. They were then panned by phage display technology. By means of Dot immunoblotting and ELISA, the libraries were identified and the Fab phage antibodies binding with antigens of colorectal cancer were selected.

RESULTS: The amplified fragments of Fd and κ gained by RT-PCR were about 650 bp. Fd and κ PCR products were subsequently inserted into the vector pComb3, resulting in a recombination rate of 40% and the volume of Fab phage display library reached 1.48 × 10⁶. The libraries were enriched about 120-fold by 3 cycles of adsorption-elution-multiplication (panning). Dot immunoblotting showed Fab expressions on the phage libraries and ELISA showed 5 clones of Fab phage anti bodies which had binding activities with antigens of colorectal cancer.

CONCLUSION: The natural immune Fab antibody phage display libraries of colorectal cancer were constructed. They could be used to select the relative antibodies of colorectal cancer.

Key Words: colorectal neoplasms, immunology, bacterio phages, antibody library

INTRODUCTION

The incidence of colorectal cancer is growing in China. It has been clear that the prognosis of colorectal cancer is related to early diagnosis and treatment[1-15]. Since the development and evolution of the colorectal cancer is one kind of complicated procedures involving multi-genes, multi-factors and multi-steps, there have been no highly peculiar molecular pathologic changes and tumor[16-39]. A major focus of cancer immunology is on the isolation of antibodies that react selectively with human tumor cells[40,41], because the antibodies could have important applications for targeting diagnostic and therapeutic agents to tumors and for identifying tumorigenic antigens[42-47]. The established approach has been to generate large panels of monoclonal antibodies (mAb) from mice immunized with human tumor cells and to screen the antibodies for reactivity against the tumor[48-50]. Despite the enormous efforts put on this approach, few antibodies that react preferentially with human tumors, and none that react specifically with one type of tumor (such as colorectal cancer), have been reported. Further attempts to isolate more specific and high affinity antibodies will require improved methods of generating and selecting antibodies against human tumors[51]. One is the introduction of method for synthesizing virtually the entire repertoire of a person’s antibody genes of variable regions by PCR technique and for expressing the encoded antibodies on the surface of a phage vector[52]. The resulting phage antibody library can be panned to select and clone rare antibodies on the basis of their binding specificities[53,54]. It can resolve the problems of generating humanization mAb by hybridoma approach. Humans can be immunized or are immune to many antigens (including tumor antigens) but only local lymph nodes, a productive source of antibody-producing cells, is readily available. Here we describe the construction of the natural immune phage display libraries expressing Fab antibodies derived from local cancer metastasis lymph nodes of co lorectal cancer patients and report the initial results of panning the libraries for anti-colorectal cancer antibodies.

MATERIALS AND METHODS

Vector, E. coli and helper phage

The vector (pComb3), about 4894 base pairs, contains ampicillin resistance (Ampr) gene, start sites of plasmid CO1E1 and f1 phage replication. The expression of inserting fragments is controlled by the same LacZ promoter on its upstream sequence. E. coli XL1-Blue contains tetracycline resistance (Tetr) gene on its gene type of Tn10. Helper phage (VCSM13) contains kanamycin resistance (Kanar) gene with valency of 10¹⁵ pfu•L¯¹. It is amplified in SOC culture medium and preserved in 4 °C.

Lymph nodes total RNA preparation

Lymph nodes in mesenterium were resected during surgical operation on patients with colorectal cancer and preserved in li quid nitrogen immediately. The nodes of patients (case number 260280, 260583 and 260476) were defined as tumor metastatic lymph nodes by pathological examination. One hundred mg of each node was used to extract total RNA by the standard method of guanidinium isothiocyanate.

Amplifying Fd and κ chain genes of antibodies by RT-PCR

Total RNA (20-50 μg) was added to 60 pmol primer of Oligo (dt) and heated at 65 °C for 10 min. The mixture was then used in a 20 μL reverse transcription reaction containing 200 μmol•L¯¹ each dNTPs and 20 U of reverse transcriptase (Promega), which was incubated at 37 °C for 1 h. The RNA-cDNA mixture (5 μL) was then used in 50 μL PCR reaction mixture containing all four dNTPs at 60 μmol•L¯¹, 5 U of Taq polymerase (Promega), and 50 pmol•L¯¹ of appropriate 5’ and 3’ primers[55,56]. VK1a and VK3a are 5’ primers for amplification of the κ chain with the Sac I site for cloning into the vector pComb3. CK1a is a 3’ primer corresponding to the 3’ end of the light chain κ, Xba I site. VH1a and VH3a are 5’ primers for the heavy chain (Fd), Xho I site. CG1z is the 3’ primer for the Fd and corresponds to part of the hinge region, Spe I site.

V_κ5’ primers: VK1a, 5’-GACATCGAGCTCACCCAGTCTCCA -3’;

V_κ3a, 5’-GAAATTGAGCTCACGCAGTCTCCA-3’;

V_κ3’ primers: CK1a, 5’-GCGCCGTCTAGAACTAACACTCTCCCCTGTTGAAGCTCTTTGTGACGGGCAAG-3’;

V_H (Fd) 5’ primers: VH1a, 5’-CAGGTGCAGCTCGAGCAGTCTGGG-3’;

V_H3a, 5’-GAGGTGCAGCTCGAGGAGTCTGGG-3’;

V_H (Fd) 3’ primers: CG1z, 5’-GCATGTACTAGTTTTGTCACAAGATTTGGG -3’.

The reaction mixtures were then subjected to 35 rounds of amplification (PE/Cetus thermal cycler) at 94 °C for 1 min, 52 °C for 1.5 min and 72 °C for 2 min followed by a final incubation at 72 °C for 10 min. An aliquot of the reaction mixture (5 μL) was run on a 10 g•L¯¹ agarose gel.

Cloning heavy chain Fd into pComb3

The Fd fragment product of PCR (isolated by agarose gel electrophore sis) was cut with an excess of the restriction enzymes Xho I and Spe I and typically about 350 ng was ligated with 2 μg of Xho/Spe I -linearized pComb3 vector (isolated by agarose gel electrophoresis) in a total volume of 150 μL with 10 U of ligase (Promega) at 16 °C for 15 h. Following ligation, DNA was precipitated at -20 °C for 2 h by the addition of 15 μL of 3 mol•L¯¹ sodium acetate (pH5.2), and 330 μL of ethanol. DNA was pelleted by microcentrifugation at 4 °C for 15 min. The DNA pellet was resuspended in 20 μL of water and transformed into 150 μL XL1-blue (porated by calcium chloride). After transformation, XL1-blue samples (10, 1, 0.1 μL) were withdrawn for plating to determine the rate of transformation. The insertion of target Fd fragments were detected by PC R from the plasmids extracted from several random XL1-blue monoclones. The plasmids with Fd insertion were named p+Fd.

Cloning light chain κ into p+Fd and antibodies Fab libraries construction

The κ fragment product of PCR and recombined p+Fd (isolated by agarose gel electrophoresis) were cut with an excess of the restriction enzymes Sac I and Xba I. The ligation, transformation, E. coli amplification and determination of the transformation rate were same as described above. The insertion of target κ fragments was detected by digestion with Sac I/Xba I from the plasmids extracted from several random XL1-blue monoclones. The Fab fragments’ insertion was detected by digestion with Xho I and Xba I. The plasmids with Fd together with κ insertion were named Fd+κ. Helper phage VCSM13 (10¹² pfu) was added to XL1-blue samples contained Fab gene libraries. Following the superinfection, the primer Fab phage display libraries of two patients were constructed, named K1 and K2, and preserved at 4 °C.

Panning of the primer Fab phage display libraries

The colorectal cancer antigens were derived from fresh cancer tissues of patients 1, 2, and 3 by sonicating the tissues (each mass 500 mg). The supernatants were named Ag1, Ag2 and Ag3. Colon cancer cell line of Lovo (10⁹•L¯¹) was sonicated also, and its supernatants were named Lovo. The wells of a microtiter plate were coated overnight at 4 °C with the antigen supernatants above respectively (antigens were diluted with 0.1 mol•L¯¹ bicarbonate buffer, pH9.6). Cancer tissue antigens coated the microtiter plate wells in mixture of Ag1 and Ag2. Lovo cells of 200 μL (0.5 × 10⁸•L¯¹) were attached to the wall of the microtiter plate wells, and 50 μL mixture of K1 and K2 was added to the wells coated with antigens respectively. The panning procedure is a modification[57] of that originally described by Parmley and Smith. The phage binding with antigens re-infected the XL1-Blue by eluting from the wells. A round of panning was finished after superinfection by VCSM13. Following 3 rounds of panning, the percent yield of phage was determined as (no. of phage eluted/no. of phage applied) × 100.

Dot immunoblotting analysis of Fab displaying[58]

Five μL library of each round of panning was added to nitrocellulose (NC) filter (with diameter of 0.5 cm). After being dried at room temperature, the NC filters were blocked in 50 mL•L¯¹ de-lipid milk and then react with the biotinylated anti-human IgG (1:100 dilution) and alkaline phosphatase (AP)-avidin at room temperature successively. Finally the NBT+BCIP was added and color developed. The control unit was the suspension of the phage contained vacant vector pComb3.

ELISA analysis of Fab displaying[59]

The wells of a microtiter plate were coated overnight at 4 °C with the antigen supernatants Ag1, Ag2, Ag3 and Lovo respectively (antigens were diluted in 1:10 0 with 0.1 mol•L¯¹ bicarbonate buffer, pH9.6). The suspension of K1 and K2 (50 μL each) was added to the wells respectively, and cultivated at 37 °C. Following 3 washes with TBS buffer, 25 μL of a 1:100 dilution of biotinylated goat anti-human IgG and HP-conjugated biotin were added successively and incubated at 37 °C. Finally 50 μL of TMB was added and color development was monitored at 490 nm. The A 490 values of positive clones were higher than that of negative clones at least two folds. After 3 rounds of panning, 5 clones of positive XL1-blue were superinfected with VCSM13 and the supernatants of Fab displaying were prepared. Fifty μL of the Fab display supernatants were added to the wells (coated with antigens of Ag1, Ag2, Ag3 and Lovo) respectively, and ELI SA analysis was applied by the method described above.

RESULTS

Amplifying the fragments of Fd and κ chain genes of antibodies by RT-PCR

The total RNAs of the lymph nodes defined as tumor metastasis in mesenterium of patients with colorectal cancer were isolated. The integrity of RNAs was shown by alkaline denaturing agarose gel. Their purity (A₂₆₀/A₂₈₀) reached 2.04. Fd and κ immunoglobulin chains were PCR-amplified after reverse transcription from this RNA. For amplifying Fd and κ, two 5’primers and one 3’primer were used respectively which contained some sites of the restriction enzymes corresponding to the vector pCom b3. The PCR amplification of Fd and κ was about 650 bp (Figure 1).

Figure 1 PCR products of Fd and κ.

Construction and identification of recombinant p+Fd

The recombinants of p+Fd were constructed by cloning Fd fragments into pComb3. After transforming XL1-blue, the Fd gene libraries were formed. And the quantity of the transformants reached 5.53 × 10⁶. After extracting the plasmids from eight random XL1-blue monoclones, the insertion of target Fd fragments was detected by PCR with the primers same as that in amplifying Fd described above. Four of eight PCR could amplify the fragments of 650 bp (Figure 2). This meant that the recombination fre quency was 50%, so the practical volume of Fd library was 2.77 × 10⁶.

Figure 2 Fd insertion by PCR identification.

Construction and identification of Fab gene recombinant

The recombinants of Fd+κ were constructed by cloning κ fragments into p+Fd. After transforming XL1-blue, the Fab gene libraries were formed and the quantity of the transformants reached 7.4 × 10⁶. Following extracting the plasmids from ten random XL1-blue monoclones, the insertion of target κ fragments was detected by digestion with Sac I and Xba I, and five of these plasmids discharged the fragments of 650 bp (Figure 3). By digesting with Xho I and Xba I, four of these plasmids discharged the fragments about 2.4 kb (Figure 4). The recombination frequency of fragments Fd together with κ insertion was 40%, so the practical volume of Fab library was 7.4 × 10⁶× 50% × 40% = 1.48 × 10⁶.

Figure 3 Identification of κ insertion by Sac I/Xba I digestion.

Figure 4 Identification of κ and Fd insertion by Xho I/Xba I digestion.

ELISA of binding activity of colorectal cancer antigens and Fab displaying on primer phage libraries

The activity of colorectal cancer antigens binding with K1 (or K2) was identified by ways of ELISA with control unit of wash buffer. The primer phage display libraries integrated with the colorectal cancer antigens of not only its immune derivation, but also other immune derivation (Table 1).

Table 1 Binding of Fab displayed by prime phage display librar ies with colorectal cancer antigens (A₄₉₀).

	Ag1	Ag2	Ag3	Lovo	Wash buffer
K1	1.285	1.496	1.332	0.326	0.105
K2	1.324	1.552	1.352	0.322	0.122
BC	0.115	0.105	0.110	0.092	0.087

Panning and identification of the Fab phage display libraries

After 3 rounds of panning, the libraries were enriched about 120-fold. Dot immunoblotting applied by the system of AP-goat anti-human IgG showed that there were Fab expressions on the phage libraries of each panning round (Figure 5). There was no obvious color development in the control units (C). The supernatants of Fab displaying prepared from 5 clones of positive XL1-Blue after 3 rounds of panning showed the significant binding activity with the antigens related with colorectal cancer by ELISA analysis (Table 2). The control units were the rat colon antigens and wash buffer.

Table 2 Binding activity of phage antibodies with antigens (A₄₉₀）.

Phage antibodies	Ag1, Ag2 and Ag3	Lovo	Rat colon antigens	Wash buffer
Clone 1	1.236	1.042	0.121	0.096
Clone 2	1.117	0.873	0.110	0.100
Clone 3	1.450	1.329	0.182	0.121
Clone 4	1.106	0.912	0.106	0.087
Clone 5	1.345	1.024	0.090

Figure 5 Dot immunoblotting analysis.

DISCUSSION

Fd parts and κ immunoglobulin chains (650 bp) were PCR-amplified after reverse transcription from the RNAs, which were isolated from the lymph nodes defined as tumor metastasis in mesenterium of patients with colorectal cancer. For mimicking the diversity of the antibody genes, we used 5’ primers based on the leader exon and the N terminus of first framework (FR1) sequences according to their relatively constant regions, and 3’ primers within the constant regions during amplifying the V genes of immunoglobulin Fab. To maximize complementarity, degeneracy was incorporated into the primers, or different primers were designed for different families of antibody V genes. For cloning of the amplified DNA into expression vectors, rare restriction sites were introduced within the PCR primer s. Following mixing the 2-group of Fd PCR products, we cloned them into the vector pComb3. Then the 2-group of κPCR products were inserted into the recombinants of p+Fd. Therefore the gene libraries of humanization antibody were constructed, which contained parts of 2-subgroup of heavy chains and 2-subgroup of κ chains. Previously reports had shown that the volume of the phage display libraries was 1.0 × 10⁷ at most constructed by this vectors and ways[57]. In review, the volume was 10⁶～10⁷ according to the reports abroad, and about 10 6 according to the domestic reports[60,61]. We reported the volume being 1.48 × 10⁶.

By antibody technology, the mAbs can be prepared by-passing hybridoma approach. Construction of the natural immune antibody library is the method of by-passing unnatural immunization. The natural immune antibody library is constructed from the natural donors, such as lymphocyte B and plasma cell. In our present study, the natural immune Fab antibody phage display libraries were constructed from the lymphocytes in the lymph nodes of 2 patients with colorectal cancer. There have been several studies on the library of immunity antibody in recent years[60-62], but no reports on construction of the natural immune antibody library of human colorectal cancer. Theoretically the humanization antibody library constructed from the lymphocytes B in lymph nodes defined as tumor metastasis of patients with tumor can aim at screening the antibodies, because the genes of heavy and light chains have been rearranged and ligated specifically due to immunizing of tumor antigens. By this way the promiscuous work of preparation of antigens by immunizing rats was relieved and the problems of weak antigens, heterological antibodies, low fusion, instability and low antibodies products of hybridoma were resolved. From the natural immune antibody library, a few of phage antibodies immunized by some for eign antigens (such as bovine serum album, lysozyme, bovine thyroid globulin, etc.) were screened, and some self-antigens (such as tumor necrosis factor-α, carcinoembryonic antigen, CD4, thyroid globulin, etc.). Many of these phage antibodies showed high affinity and specificity, and the potential value for application.

It is important to assemble antibody molecules that have correctly folded heavy and light chains and thus retain antigen-recognition capabilities. Previously it had been demonstrated that Fd chains (comprising V_H and C_H1, the variable region and constant domain 1 of the immunoglobulin heavy chain) and κ chains targeted to the periplasm of E. coli assemble to form functional Fab molecules. If the Fd chain was anchored in the membrane and concomitantly provided with secreted κ chain, functional Fab molecules could form on the membrane surface facing the periplasm. The coexpression behind the PelB leader sequence of Fd fused to cpIII (the major coat protein of M13 phage) and κ chains lead to membrane anchoring of the Fd chain and compartmentalization of the κ chains in the periplasm. These two chains could assemble in situ to allow accumulation of functional Fab on the membrane surface, which, by virtue of the cpIII sequences, would be incorporated along the entire length of the filamentous phage particles on subsequent infection with helper phage. These phages displaying Fab could infect E. coli again. Following rounds of panning by adsorption-wash-amplification, the phages comprising specific antibody molecules could be enriched[63]. Our study showed that the phage Fab libraries were enriched about 120-fold after 3 rounds of panning. It suggested that the colorectal cancer antigens had selected the phages in the libraries displaying Fab. Dot immunoblotting showed that there were Fab expressions on the phage libraries of each panning round. ELISA analysis showed that the phage displaying Fab had the significant binding activity with the antigens related with colorectal cancer. Considering that nowadays there are few and hypo-specific antibodies related with colorectal cancer, the natural immune Fab antibody phage display libraries of colorectal can be used to select the affinity and specificity antibodies related to colorectal cancer. It is important to diagnose and treat colorectal cancer using these antibodies.