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. 2001 Feb 13;98(4):1799-804.
doi: 10.1073/pnas.98.4.1799.

A human myeloma cell line suitable for the generation of human monoclonal antibodies

A Karpas  1 , A Dremucheva, B H Czepulkowski
Affiliations

A human myeloma cell line suitable for the generation of human monoclonal antibodies

A Karpas et al. Proc Natl Acad Sci U S A. .

Abstract

Ever since monoclonal antibodies were produced in 1975 with mouse myeloma cells there has been interest in developing human myeloma cultures for the production of monoclonal antibodies. However, despite multiple attempts, no human myeloma line suitable for hybridoma production has been described. Here we report the derivation of a hypoxanthine-aminopterin-thymidine-sensitive and ouabain-resistant human myeloma cell line (Karpas 707H) that contains unique genetic markers. We show that this line is useful for the generation of stable human hybridomas. It can easily be fused with ouabain-sensitive Epstein-Barr virus-transformed cells as well as with fresh tonsil and blood lymphocytes, giving rise to stable hybrids that continuously secrete very large quantities of human immunoglobulins. The derived hybrids do not lose immunoglobulin secretion over many months of continuous growth. The availability of this cell line should enable the in vitro immortalization of human antibody-producing B cells that are formed in vivo. The monoclonal antibodies produced may have advantages in immunotherapy.

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Figures

Figure 1
Figure 1
Analysis of Ig secreted by the Karpas 707H cell line and hybridomas. The cells were grown at a concentration of 2 × 106 cells per milliliter in l-methionine, l-cysteine-deficient medium that was supplemented with 10% dialyzed FBS and with [35S]methionine and [35S]cysteine at 250 μCi/ml (Amersham Pharmacia). The cells were incubated for 8 h at 37°C in a CO2 incubator. After incubation, the cell suspensions were centrifuged at 1,000 × g for 5 min. The supernatant was analyzed by SDS-PAGE after total reduction with 10% Bis-Tris Gel with Mops running buffer (Invitrogen NuPage Electrophoresis system). Tracks: 1, small quantity of light chain produced by the Karpas 707H cells; 2, IgG that reacts with the gp41 HIV-1 produced by the hybrid with the EBV-infected 164 cells; 3 and 4, IgG-producing hybridoma formed with fresh WBC; 5–9 and 13–16, hybridoma formed with tonsil cells. Track 10 may contain both IgG and IgM, probably because of a mixture of two hybridomas that were formed in the same well. Tracks 11 and 12, IgM-secreting hybridomas; tracks 12–14, hybridomas that secrete two distinct light chains. This figure illustrates that in such hybridomas the secretion of the myeloma λ light chain is greatly amplified compared with the nonfused myeloma (track 1). What appears to be a single line of light chains in most hybridomas is probably due to myeloma and donor chains banding in the same position.
Figure 2
Figure 2
Quantitation of IgG secretion by two hybridomas: 707H/164 (○) and 707H/100 (●). Each hybridoma was seeded at a concentration of 3 × 105 cells per milliliter in RPMI 1640 growth medium supplemented with 10% FBS. Samples were collected at the indicated intervals for IgG quantitation by the nephelometric assay and for cell counts. After 4 days the viable cell count of the 707H/164 hybridoma reached 8 × 105 cells per milliliter, whereas that of a 707H/100 reached 6 × 105 cells per milliliter. Thereafter the number of viable cells decreased, and by the 10th day nearly all of the cells were dead when the level of IgG in the medium was at its highest, namely 210 μg/ml of IgG for both cultures.
Figure 3
Figure 3
(A) A karyotype of the chromosomes of the Karpas 707H cell line, which are near-tetraploid. Note that chromosomes 2, 14, and 22, which contain the Ig genes, are normal and diploid. 83,XX,-X,-X,del(1)(p11p36)x4,+del(1)(q11q44),-2,-2,der(3)del(3)(p21p25)inv(3) (q23q25),+del(3)(q13q21),der(5)t(1;5)(p11;q31),der(5)t(2;5)(q21;q31), del(5)(q13q33)x2,+add(5)(q31),-6,-6,add(7)(p25),del(8)(q22q24),-9, del(11)(q23q25),-11,del(12)(p12p13),-13,-13,-14,-14,add(15)(p11)x2, der(16)t(1;16)(p11;p11),add(16)(p11)x2,-16,der(18)t(1;18)(q23;p11),add(18)(p11),add(18)(q23),-18,add(19)(q13)x2,+der(19)t(1;19)(q11;p13) +der(19)t(dup(1)(q23q44);19)(q11;p13),-21,-22,-22,+mar1 × 2,+mar2. (B) A karyotype of the hybridoma Karpas 707H/164, which is near-hexaploid, containing the normal pairs of chromosomes that are readily obvious in the case of chromosomes no. 1. 125,XXX,-X,-X,-X,del(1)(p11p36)x4,+del(1)(q11q44),-2,-2,add(3)(p25),der(3)del(3)(p21p25)inv(3)(q23q25),add(4)(q31),-4,der(5)t(1;5)(p11;q31), der(5)t(2;5)(q21;q31),del(5)(q13q33)x2,+add(5)(q31),del(6)(q21q23) -6, -6,add(7)(p25)x2,del(8)(q22q24),-9,del(11)(q23q25),-11, del(12)(p12p13)x2,-13,-13,-14,-14,add(15)(p11)x2,der(16)t(1;16)(p11;p11), add(16)(p11)x2, -16,der(18)t(1;18)(q23;p11),add(18)(p11),add(18)(q23), -18,add(19)(q13)x2,+der(19)t(1;19)(q11;p13),+der(19)t(dup(1)(q23q44);19)(q11;p13), -21,-21,-22, -22,+mar1 × 2,+mar2.
Figure 4
Figure 4
Diagram of the abnormal chromosomes found in the Karpas 707H myeloma and hybridoma. For comparison a normal chromosome is also illustrated with the corresponding abnormal chromosomes.
Figure 5
Figure 5
Electron micrographs of the myeloma (A), 164 lymphoblast (B), and hybridoma that produces monoclonal antibodies against HIV (C). (×4,500.) The hybridoma shows extensive RER. The amount of RER in the cytoplasm of hybridoma derived with WBC was very similar to that shown in C.
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References

    1. Cotton R G H, Milstein C. Nature (London) 1973;244:42–43. - PubMed
    1. Köhler G, Milstein C. Nature (London) 1975;256:495–497. - PubMed
    1. Gustafsson B, Hinkula J. Hum Antibod Hybridomas. 1994;5:98–104. - PubMed
    1. Lloyd-Evans P, Gilmour J E M. Hybridoma. 2000;19:143–149. - PubMed
    1. Nilsson K. Int J Cancer. 1971;7:380–396. - PubMed

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