Mammalian cell lines used in antibody engineering and expression system

1. Mammalian cell lines

1.1. Mouse myeloma and hybridoma cell lines derived from MOPC21 and used as fusion partners

Among the cell lines derived from MOPC21, the SP2/0 (hybridoma), P3-X63-Ag8.653 (myeloma) and NS0 (myeloma) cell lines are frequently used as fusion partners in the generation of hybridomas. The table below shows their clone origin and their characteristics.

Cell line name	Other cell line names	Species and strain	Cell type and cell lineage	Clone origin	IG production	Cell line characteristics	ECACC	ATCC	IMGT references
MOPC21	MOPC 21, MOPC21 (mineral oil induced plasmacytoma number 21)	Mus musculus BALB/c (inbred female mouse)	myeloma, B cell		secreting IgG1-kappa (H+L+)				Horibata and Harris 1970 [1]
P3K		Mus musculus BALB/c	myeloma, B cell	derived from MOPC21	secreting IgG1-kappa (H+L+)	suspension, heterogeneous population			Horibata and Harris 1970 [1]
P3-X27		Mus musculus BALB/c	myeloma, B cell	cloned from P3K	secreting IgG1-kappa (H+L+)				Ramasamy et al. 1974 [2]
NSI/1	289-16	Mus musculus BALB/c	myeloma, B cell	cloned from P3-X27	non secreting, synthesing kappa (H-L+ intracellular)	suspension			Ramasamy et al. 1974 [2], Cowan et al. 1974 [7]
NS-1	P3/NSI/1-Ag4-1, NS-1-Ag4-1, P3-NS-1/1-Ag4-1	Mus musculus BALB/c	myeloma, B cell	cloned from NSI/1	non secreting, synthesing kappa (H-L+ intracellular)	suspension, resistant to 8-azaguanine	85011427	TIB-18™	Köhler et al. 1976 [8]
NS0	NS/0, NS0/1	Mus musculus BALB/c	myeloma, B cell	cloned from NS-1	non-IG secreting, non-IG synthetising (H-L-)	suspension, resistant to 8-azaguanine	85110503		Galfrè and Milstein 1981 [9]
P3-X63		Mus musculus BALB/c	myeloma, B cell	cloned from P3X27	secreting IgG1-kappa (H+L+)				Ramasamy et al. 1974 [2]
P3-X63-Ag8	P3X63Ag8, P3/X63Ag8, X63	Mus musculus BALB/c	myeloma, B cell	cloned from P3X27	secreting IgG1-kappa (H+L+)	suspension, resistant to 8-azaguanine	85011401	TIB-9™	Köhler et Milstein 1975 [3]
P3-X63-Ag8-U1	P3X63Ag8U.1, P3X63Ag8U1, P3U1	Mus musculus BALB/c	myeloma, B cell	variant of P3-X63-Ag8.653	non-IG secreting, synthesing kappa (H-L+)	suspension	94120802	CRL-1597™	Yelton D.E. et al. 1979 [5]
P3-X63-Ag8.653	P3X63Ag8.653, P3/X63Ag8.653, X63-Ag8.653, X63Ag8.653, X63.653, Ag8	Mus musculus BALB/c	myeloma, B cell	cloned from P3-X63-Ag8	non-IG secreting, non-IG synthetising (H-L-)	suspension	85011420	CRL-1580™	Kearney J.F. et al. 1979 [6]
Sp2/0	Sp2/0-Ag-14, Sp2/0-Ag14, SP-2	Mus musculus BALB/c	hybridoma, B cell	obtained by fusion with P3X63Ag8	non-IG secreting, non-IG synthetising (H-L-)	suspension, resistant to 8-azaguanine	85072401	CRL-1581™ CRL-8287™	Shulman M. et al. 1978 [4]
YB2/0	YB2/3HL.P2.G11.16Ag.20	Rattus norvegicus	hybridoma, B lymphoblast	obtained by fusion with Y3/Ag1.2.3	non-IG secreting	suspension, resistant to 8-azaguanine		CRL-1662™	Kilmartin JV, et al. 1982 [10]

YB2/0 was obtained by fusing Y3/Ag1.2.3 (ATCC CRL-1631) cells with spleen cells from an AO strain rat. YB2/0 can be used as a fusion partner for rat B cells to make rat - rat hybridomas.

1.2. Rat hybridoma cell line used as fusion partner

Cell line	Other cell line names	Species and strain	Cell type and cell lineage	Clone origin	IG production	Cell line characteristics	ECACC	ATCC	IMGT references
YB2/0	YB2/3HL.P2.G11.16Ag.20	Rattus norvegicus	hybridoma, B lymphoblast	obtained by fusion with Y3/Ag1.2.3	non-IG secreting	suspension, resistant to 8-azaguanine		CRL-1662™	Kilmartin JV, et al. 1982 [10]

The cell lines used as fusion partners do not grow in HAT medium (they die in the presence of HAT, they are HAT or HAz-sensitive). They lack a functional HGPRT, i.e., they are resistant to 8-azaguanine (the Ag in the cell names stands azaguanine).

Cholesterol requirements
NS-1, P3-X63-Ag8, P3-X63-Ag.653 are cholesterol auxotrophs (requires cholesterol). In contrast, hybridoma Sp2/0 do not require neither LDL nor cholesterol (the fusion partner bringing the necessary genes) [11].

Figure 1. Myeloma cell lines derived from MOPC21 showing SP2/0 (hybridoma), P3-X63-Ag8.653 (myeloma) and NS0 (myeloma) used as fusion partners in the generation of hybridomas.

The original hybridoma Sp2/3-3 was obtained by fusion of BALB/c spleen cells producing an anti-SRBC mAb Sp2 (from mouse immunized with sheep RBC) with the P3-X63-Ag8 (X63) myeloma [3].

1.3. Hamster cell lines used as protein expression system

Cell line	Other cell line names	Species and strain	Cell type and cell lineage	Clone origin	IG production	Cell line characteristics	ECACC	ATCC	Companies	IMGT references
CHO	CHO-ori, Chinese Hamster Ovary	Cricetulus griseus	epithelial			adherent	85050302			Puck TT et al. 1958 [12]
FreeStyle™ CHO-S Cells	CHO-derived suspension culture	Cricetulus griseus		derived from CHO-S		suspension			Life Technologies:R800-07
CHO-K1	CHOK1, CHO K1, CHO cell clone K1	Cricetulus griseus	epithelial	derived from CHO		adherent	85051005	CCL-61™		Kao FT and Puck TT 1968 [13]
POTELLIGENT® CHOK1SV	CHO-K1SV, suspension variant of CHO-K1	Cricetulus griseus		derived from CHO-K1		suspension			Lonza
CHO/dhFr-		Cricetulus griseus	epithelial			adherent, dhFr-	94060607
CHO-DUK-B11	CHO duk-, DXB11, CHO-DUKX	Cricetulus griseus	epithelial	derived from CHO-K1		adherent, dhFr-		CRL-9096™		Urlaub G et al. 1980 [14]
CHO-DG44 Cells (cGMP banked)	CHO DG44	Cricetulus griseus		derived from CHO-K1		suspension, dhFr-			Life Technologies:A11000-01	Urlaub G et al. 1983 [15]

1.4. Human cell lines

HEK293 cell line derived from human embryonic kidney cells transformed with sheared fragments of adenovirus type 5 DNA has been widely used in the production of monoclonal antibody. Some derivatives were further transformed either with the simian virus 40 (SV40) large T antigen, termed HEK293T, or with the Epstein Barr virus (EBV) nuclear antigen 1 (EBNA1), termed HEK293E, using an origin of replication (ori) of SV40 or EBV, respectively.

PER.C6® cell lines are derived from human embryonic retina cells that have been immortalized by transfecting the E1 genes from adenovirus 5 DNA.19. PER.C6® cells can proliferate indefinitely in suspension under serum-free conditions.

2. Selection

2.1. HAT

Hybridoma cells are selected using HAT (Hypoxanthine Aminopterin Thymidine) medium.

HAT selection depends on the fact that mammalian cells can synthesize nucleotides by two different pathways: the de novo and the salvage pathways. Aminopterin (a folic acid analog) blocks the de novo pathway, while hypoxanthine and thymidine allow growth via the salvage pathway.

Indeed, the DNA de novo synthesis (in which a methyl or formyl group is transferred from an activated from of tetrahydrofolate) is blocked by Aminopterin. When the de novo pathway is blocked, cells utilize the salvage pathway, which bypasses the aminopterin block by converting purines and pyrimidines directly into DNA. The enzymes catalyzing the salvage pathway include hypoxanthine-guanine phosphorribosyl transferase (HGPRT) and thymidine kinase (TK). This pathway can convert hypoxanthine in IMP, a reaction catalysed by HGPRT. It can also convert thymidine in dTMP, a reaction catalysed by TK.

On this medium, only cells which have functional HGPRT and TK will grow via the salvage pathway.



Figure: Principle of HAT selection (origin: http://nfs.unipv.it/nfs/minf/dispense/immunology/lectures/files/monoclonal_antibodies.html)

More information: Monoclonal antibodies are commonly assumed to be monospecific, but anecdotal studies have reported genetic diversity in antibody heavy chain and light chain genes found within individual hybridomas. In order to evaluate the prevalence of such diversity, 185 random hybridomas were analysed in a large multicenter dataset [16]. Bradbury ARM, Trinklein ND, Thie H, Wilkinson IC, Tandon AK, Anderson S, Bladen CL, Jones B, Aldred SF, Bestagno M, Burrone O, Maynard J, Ferrara F, Trimmer JS, Görnemann J, Glanville J, Wolf P, Frenzel A, Wong J, Koh XY, Eng HY, Lane D, Lefranc M-P, Clark M, Dübel S. When monoclonal antibodies are not monospecific: hybridomas frequently express additional functional variable regions MAbs. 2018 May/Jun;10(4):539-546.doi: 10.1080/19420862.2018.1445456. Epub 2018 Mar 29.PMID: 29485921 Free PMC Article.