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Cloning, Expression, and Modification of Antibody V Regions

Sherie L. Morrison¹

¹University of California, Los Angeles, California

Publication Name:

Current Protocols in Immunology

Unit Number:

Unit 2.12

DOI:

10.1002/0471142735.im0212s47

Online Posting Date:

May, 2002

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Abstract

Cloned variable (V) regions of antibodies can be expressed joined to any constant (C) region, from either the same or a different species. The resulting antibodies will have the desired associated effector functions. Chimeric antibodies obtained by joining murine V regions to human C regions should have decreased immunogenicity in humans. The process of complementarity determining region (CDR) grafting, in which the CDRs from an antibody of one species are transferred to the framework regions of another species, constitutes a further modification of this approach. The protocols presented in this unit are designed to permit PCR-based cloning of heavy and light chain V regions. This is an advanced molecular biology protocol and should be employed only by investigators who are sufficiently skilled and experienced.

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Unit Introduction
Basic Protocol 1: Cloning and Expression of Immunoglobulin Variable Regions Using PCR with Redundant Primers
Support Protocol: Production of TA Vector
Basic Protocol 2: ELISA to Identify Transfectomas that Secrete Antibody Molecules
Basic Protocol 3: Characterization of Antibody Molecules Produced by Transfectomas
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Cloning and Expression of Immunoglobulin Variable Regions Using PCR with Redundant Primers

Materials

5 × 10⁵ cells from antibody-producing hybridoma cell line (units 2.1 & 2.5)
Phosphate-buffered saline (PBS; appendix 2A)
Guanidinium thiocyanate denaturing solution (unit 10.11)
2 M sodium acetate, pH4 (unit 10.11)
Water-saturated phenol (unit 10.11)
49:1 (v/v) chloroform/isoamyl alcohol
100% and 70% ethanol, –20°C
Diethylpyrocarbonate (DEPC)–treated H₂O (unit 10.11)
1 mg/ml 3¢ primer for cDNA synthesis (Table 2.12.1)
First-strand cDNA buffer (see recipe)
50 U/µl RNase inhibitor (RNasin, Promega)
10 mM 4dNTP mix in H₂O (unit 10.9)
9.5 U/µl avian myeloblastosis virus (AMV) reverse transcriptase (Promega), diluted 1/10 in reverse transcriptase dilution buffer (see recipe)
20 µM leader primers for murine H and L chain V regions (Tables 2.12.2 and 2.12.3)
2 U/µl Taq DNA polymerase
1.25 mM 4dNTP mix in TE buffer, pH 7.5 (unit 10.20)
10× PCR amplification buffer (see recipe)
Mineral oil
Chloroform
TA vector (see Support Protocol; also available from Invitrogen)
2× T4 DNA ligase buffer (unit 10.19A)
1 Weiss U/µl T4 DNA ligase (Life Technologies)
Competent E. coli cells (appendix 3N)
20 µM J region primers (Table 2.12.4)
Expression vector for H and L chain V regions cloned by PCR (Figs. 2.12.2 and 2.12.3)
LB plates (appendix 3N)

Microcentrifuge, room temperature and 4°C
0.5-ml microcentrifuge tubes
16°, 42°, and 60°C water baths
Thermal cycler

Additional reagents and equipment for agarose gel electrophoresis (unit 10.4), alkaline lysis miniprep (unit 10.3), transforming E. coli (appendix 3N), DNA sequencing (unit 10.25), restriction endonuclease digestion (unit 10.8), purification of DNA restriction fragments (unit 10.5), and transformation of eukaryotic cells (unit 10.17B),

NOTE: All instruments and containers utilized in steps to must be autoclaved and rinsed with DEPC-treated water to avoid RNA degradation due to RNases. Gloves should be worn at all times. All water and salt solutions used in RNA preparation should be treated with DEPC. See unit 10.11 for additional information concerning DEPC treatment and the preparation and handling of RNA.

Table 2.12.1 3¢ Primers for cDNA Synthesis and Amplification of Antibody Sequences

Immunoglobulin region	PCR primer	Primer sequence^a

L chain C region	Oligo dT.R1.XBA.H3	5¢-GCCGGAATTCTAGAAGC(T)₁₇-3¢
L chain C region	MCAS.XBA^b	5¢-GCGTCTAGAACTGGATGGTGGGAGATGGA-3¢
H chain V region	MgC.C_H1AS^c	5¢-AGGTCTAGAA(C/T)CTCCACACACAGG(A/G) (A/G)CCAGTGGATAGAC-3¢

^a Degeneracies are indicated by alternative nucleotides at a single position. XbaI cloning site is underlined.

^b Designed to hybridize to sequences coding for amino acids 122 to 116 of the murine C region.

^c Antisense primer designed to hybridize to sequences coding for amino acids 130 to 120 in C_H1 of all murine immunoglobulins except IgG3.

Table 2.12.2 5¢ Sense Primers for Amplification of the Leader Region of Murine H Chain V Regions

PCR primer^a	Primer sequence^b

MHALT1.RV	5¢-GGGGATATCCACCATGG(A/G)ATG(C/G)AGCTG(T/G)GT(C/A)AT(C/G)CTCTT-3¢
MHALT2.RV	5¢-GGGGATATCCACCATG(A/G)ACTTCGGG(T/C)TGAGCT(T/G)GGTTTT-3¢
MHALT3.RV	5¢-GGGGATATCCACCATGGCTGTCTTGGGGCTGCTCTTCT-3¢
MHALT4.RV	5¢-GGGGATATCCACCATGG(A/G)CAG(G/A)CTTAC(T/A)T(C/T)(T/C)-3¢

^a3¢primers designed to hybridize to the amino terminus of H chain leader sequences. All primers are used simultaneously to amplify unknown sequences.

^bEcoRV sites (underlined) are protected by the three 5¢ Gs. Ribosome binding sites are indicated in bold. Degeneracies are indicated by alternative nucleotides at a single position.

Table 2.12.3 5¢ Sense Primers for Amplification of Murine L Chain V Regions

PCR primer^a	Primer sequence^b

MLALT1.RV	5¢-GGGGATATCCACCATGGAGACAGACACACTCCTGCTAT-3¢
MLALT2.RV	5¢-GGGGATATCCACCATGGATTTTCAGGTGCAGATTTTCAG-3¢
MLALT3.RV	5¢-GGGGATATCCACCATG(G/A)AGTCACA(G/T)AC(T/C)CAGGTCTT(T/C)(G/A)TA-3¢
MLALT4.RV	5¢-GGGGATATCCACCATGAGG(G/T)CCCC(A/T)GCTCAG(C/T)T(C/T)CT(T/G)GG(G/A)-3¢
MLALT5.RV	5¢-GGGGATATCCACCATGAAGTTGCCTGTTAGGCTGTTG-3¢
MLALT6.RV	5¢-GGGGATATCCACCATGATGAGTCCTGCCCAGTTCC-3¢

^aFive primers designed to hybridize to the amino terminus of murine L chain leader sequences. All primers are used simultaneously to amplify unknown sequences.

^b EcoRV sites (underlined) are protected by the three 5¢ Gs. Ribosome binding sites are indicated in bold. Degeneracies are indicated by alternative nucleotides at a single position.

Table 2.12.4 J Region Primers for Amplification and Cloning of Murine V Regions

PCR primer	Primer sequence^a

H chain J region^b
J_H1	5¢-GGGGCTAGCTGAGGAGACGGTGACCGTGGT-3¢
J_H2	5¢-GGGGCTAGCTGAGGATACGGGAACCGTGGT-3¢
J_H3	5¢-GGGGCTAGCTGCAGAGACAGTGACCAGAGT-3¢
J_H4	5¢-GGGGCTAGCTGAGAAGACGGTGACTGAGGT-3¢
J_H5	5¢-GGGGCTAGC TGAGGAGACTGTGACCATG-3¢
L chain J region
VLJ 1,2,4 antisense^c^d	5¢-AGCGTCGACTTACGTTT(T/G)ATTTCCA(G/A)CTT(G/T)GTCCC-3¢
VLJ 5 antisense^d^e	5¢-AGCGTCGACTTACGTTTCAGCTCCAGCTTGGTCCC-3¢
VLJ 6 antisense	5¢-AGCGTCGACTTACGTTTCAATTCCAGCTTGGTG-3¢

^a Degeneracies are indicated by alternative nucleotides at a single position.

^b NheI sites (underlined) are used for cloning into the expression vectors.

^c Primer for J1, 2, and 4; will not introduce amino acid changes.

^d SalI sites (underlined) are used for cloning into the expression vectors.

^e Primer for J5; J3 is a pseudogene.

Support Protocol: Production of TA Vector

Materials

Plasmid with a blunt-end restriction site (e.g., pBluescript from Statagene or pUC)
Appropriate restriction endonuclease (e.g., EcoRV or SmaI)
1:1 (v/v) phenol/chloroform
100% ethanol, –20°C
70% ethanol/3 M sodium acetate, pH 4.8
5× terminal transferase (TdT) buffer (see recipe)
25 mM cobalt chloride (CoCl₂)
5 mM 3¢,5¢-dideoxy-thymidine-5¢triphosphate (ddTTP)
25 U/µl terminal transferase
3 M sodium acetate, pH 4.8 (appendix 2A)
TE buffer, pH 7.5 (appendix 2A)
0.5-ml microcentrifuge tubes

Additional reagents and equipment for restriction endonuclease digestion (unit 10.8) and agarose gel electrophoresis (unit 10.4)

Basic Protocol 2: ELISA to Identify Transfectomas that Secrete Antibody Molecules

Materials

5 to 10 µg/ml goat anti-human Ig (or other appropriate antigen or antiserum) in 0.2 M carbonate buffer, pH 9.6 (do not store ³2 weeks)
Phosphate-buffered saline (PBS; appendix 2A)
Blocking buffer: 3% (w/v) bovine serum albumin (BSA) in PBS
Supernatant from growing transfectomas of interest (unit 10.17B)
Goat anti-human or chain antibody conjugated with alkaline phosphatase
Substrate: 0.6 mg/ml p-nitrophenyl phosphate in 0.9 M diethanolamine/0.24 mM MgCl₂, pH 9.8

96-well ELISA plate (e.g., Immunlon 2, Dynatech Laboratories)
Microtiter plate reader

Basic Protocol 3: Characterization of Antibody Molecules Produced by Transfectomas

Materials

1 × 10⁶ exponentially growing transfectoma cells (unit 10.17B)
Labeling medium: high-glucose Dulbeccos minimum essential medium (DMEM) deficient in methionine (Irvine Scientific)
0.01 mCi/µl [³⁵S]methionine (>1000 Ci/mmol)
NDET solution (see recipe)
Polyclonal anti-Ig (e.g., rabbit anti-Ig)
IgG Sorb (The Enzyme Center)
Sucrose pad: 30% (w/v) sucrose/0.3% (w/v) sodium dodecyl sulfate (SDS) in NDET solution (see recipe; optional)
0.3% (w/v) SDS in NDET solution (see recipe)
SDS-PAGE sample buffer (see recipe)
0.15 M 2-mercaptoethanol (2-ME)
12% Tris-glycine (Laemmli) minigels (unit 8.4)
Tris-glycine gel running buffer (unit 8.4)
Staining solution (see recipe)
Destain solution: 7% (v/v) glacial acetic acid/5% (v/v) methanol (store at room temperature)
1 M salicylate
5% phosphate gel (see recipe)
Phosphate gel running buffer (see recipe)

15-ml polystyrene tubes, sterile
IEC centrifuge (or equivalent), 4°C
Boiling water bath
Whatman 3MM filter paper

Additional reagents and equipment for polyacrylamide gel electrophoresis (unit 8.4) and autoradiography (appendix 3A)

CAUTION: This procedure should be performed only by personnel trained in the proper use of ³⁵S isotope and in NRC-licensed sites. Standard precautions to prevent excessive exposure and radioactive contamination of personnel and equipment should be followed at all times (refer to Safety Precautions for Radioisotopes, appendix 1Q) .

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Figures

Figure 2.12.1

Flow chart outlining cloning and expression of immunoglobulin V regions.

View Image
Figure 2.12.2

Vectors for the expression of heavy (H) chain variable (V) regions cloned by PCR with human gamma constant (C) regions. Filled boxes indicate the exons of the human H chain genes. Selected restriction endonuclease sites are shown, including EcoRV and NheI cloning sites within the polylinker, and arrowheads under the exons indicate the direction of transcription. Vectors have been linearized at a BamHI site; the second BamHI site 3¢ for the human sequences is shown. The PvuI site within Amp, which is usually convenient for linearizing the vectors, is indicated. The eukaryotic selectable marker is indicated by an arrow below the construct with the direction of transcription indicated. Sequences derived from pSV2 are indicated with diagonal lines. Murine noncoding sequences are indicated by a stipple pattern. The position of the promoter is indicated by light stippling. Human noncoding sequences are indicated by open boxes. The murine H chain enhancer region, located on an EcoRI fragment, is indicated by a narrow line. The EcoRV and NheI sites are used for variable region cloning. NheI and BamHI can conveniently be used to exchange constant regions. It is frequently convenient to use PvuI to linearize these plasmids.

View Image
Figure 2.12.3

Vector for the expression of light (L) chain variable (V) regions cloned by PCR. The eukaryotic selectable marker is indicated by an arrow under the sequence with the direction of transcription as indicated. Sequence origins and functions are depicted as for the H chain vectors in Figure 2.12.2. Murine V_L with the fused leader sequence is indicated by an open box. The specificity of the vector is changed by replacing the V region after cleavage with EcoRV and SalI. The direction of transcription of the immunoglobulin coding region is indicated by the arrows under the vector. The human L chain enhancer region is indicated by a narrow line. Selected restriction sites are indicated.

View Image

Literature Cited

Literature Cited
	Ausubel, F.A., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (eds.) 1994. Current Protocols in Molecular Biology. John Wiley & Sons, New York.
	Carroll, W.L., Mendel, E., and Levy, S. 1988. Hybridoma fusion cell lines contain an aberrant kappa transcript. Mol. Immunol. 25:991-995
	Coloma, M.J., Larrick, J.W., Ayala, M., and Gavilondo-Cowley, J.V. 1991. Primer design for the cloning of immunoglobulin heavy-chain leader-variable regions from mouse hybridoma cells using the PCR. BioTechniques 11:152-154.
	Coloma, M.J., Hastings, A., Wims, L.A., and Morrison, S.L. 1992. Novel vectors for the expression of antibody molecules using variable regions generated by PCR. J. Immunol. Methods 152:89-104.
	Holton, T.A. and Graham, M.W. 1990. A simple and efficient method for direct cloning of PCR products using ddT-tailed vectors. Nucl. Acids Res. 19:1156.
	Kabat, E.A., Wu, T.T., Perry, H.M., Gottesman, K.S., and Foeller, C. 1991. Sequences of Proteins of Immunological Interest, 5th ed. U.S. Department of Health and Human Services, Washington,D.C.
	Larrick, J.W., Danielsson, L., Brenner, C.A., Abrahamson, M., Fry, K.E., and Borrebaeck, C.A.K. 1989. Rapid cloning of rearranged immunoglobulin genes from human hybridoma cells using mixed primers and the polymerase chain reaction. Biochem. Biophys. Res. Commun. 160:1250-1256.