bisFabs: Tools for rapidly screening hybridoma IgGs for their activities as bispecific antibodies

Figures & data

Figure 1. Schematic diagram showing the main steps involved in producing bispecific bisFabs from murine IgGs. Purification steps are indicated in green font. The inter-chain disulfide bonds are indicated in orange. (a) The purified IgGs are digested with the proteases indicated in Table 1. to generate F(ab’)₂. (b) Reduction with TCEP releases the monomeric Fab’ molecules and reduces the heavy-light chain disulfide bond. (c) Re-oxidation of the thiol groups re-form the heavy-light disulfide bond while cyclizing a pair of cysteines in the hinge, leaving a single reduced cysteine. (d) The reoxidized Fab’ is reacted with an excess of bismaleimide crosslinker. (e) the resulting modified Fab’ is conjugated to a second Fab’ with a different specificity.

Table 1. Enzymes used to generate F(ab’)2s from the different murine isotypes.

				Fab’ product
Isotype	Enzyme	Cleagave efficiency	Main cleavage site	# of cys	abundance	bisFab yield
Mouse IgG1	Pepsin	90–100%	CKPCICTVPEVSSV↓ F	3	80–95%	130–200 μg
	Ficin	70–90%	CKPCIC↓TVP	3	90–95%	100–220 μg
Mouse IgG2a	IdeS	100%	CPPCKCPAPNLLG↓G	3	100%	190–220 μg
	Lys-C^a		CPPCKECHK↓CPA	3	10–20%
	(1:200)	90–95%	CPPCK↓ECHKCPA	2	80–90%	80–100 μg
Mouse IgG2b	Lys-C^b		CPPCKECHK↓CPA	3	70–90%
	(1:1,000)	55–65%	CPPCK↓ECHKCPA	2	10–30%	140–180 μg
Mouse IgG3	IdeS	100%	SCPPGNILG↓G	1	100%	200–220 μg

The table shows the cleavage efficiency for each enzyme in the indicated conditions as calculated from densitometry of SDS-PAGE. The major cleavage sites are indicated with the arrow (↓) in the core hinge region. # of Cys refers to the number of cysteines in the hinge region of the resulting Fab's after digestion. The relative abundance of the corresponding fragments was calculated by mass spectrometry in reducing conditions.

Bisfab yield is expressed as the amount of purified bisFabs per 1mg of starting hybridoma Ab.

Lys-C^a: using a 1:200 (wt/wt) ratio, Lys-C^b: using a 1:1,000 (wt:wt) ratio.

Figure 2. Biochemical characterization of a representative purified bisFab. (A) Non-reducing SDS-PAGE. The table indicates the structures of the corresponding bands as deduced based on the mass spectrometry analysis. The species indicated with asterisks are likely bisFabs missing one S-S bond between a heavy and light chain that dissociates under denaturing conditions. The light gray chain in the structure indicates the dissociated chain. The relative abundance of each band was determined by densitometry of 30 independent bisFabs and shown is the range observed for each band. (B) Analytical size-exclusion chromatography. The Table shows the observed range of aggregation and low molecular weight fragments obtained for the panel of bisFabs and the molar masses calculated by multi-angle laser light scattering. (C) Mass spectrometry analysis showing mass deconvolution between 20 kDa and 120 kDa, and (D), a zoom-in of the mass spectrometry analysis in the 98 kDa area. Homodimers were absent or below 2%.

Figure 3. Comparison of T-cell recruiting antibodies using 3 separate anti-HER2 antibody clones (A: 4D5, B: 2C4, C: 7C2) in either full-length IgG (blue line) or bisFab format (red line). Shown is the percentage of target cell (SKBR-3) killing in the presence of human PBMCs and different concentrations of the antibodies. Values are the mean ± standard deviation of 3 replicate experiments. The calculated EC₅₀ and extent of maximum killing are: (A) IgG: 0.24 pM, 95%; bisFab: 0.07 pM, 95%; (B) IgG: 0.14 pM, 90%; bisFab: 0.06 pM, 90%; (C) IgG: 46 pM, 65%; bisFab: 129 pM, 62%.

Figure 4. T-cell killing activity of 3 antibodies against B-cell targets (A: anti-CD20, B: anti-CD79 clone A, C: anti-CD79 clone B). Antibodies in the full-length IgG format (blue line) or bisFabs (red line) were tested against BJAB cells in an in vitro cell killing assay using human PBMCs. Values are the average of duplicate experiments ± standard deviation. The calculated EC₅₀ and extent of maximum killing are: (A) IgG: 1.9 pM, 85%; bisFab: 0.8 pM, 85%; (B) IgG: 200 pM, 80%; bisFab: 49 pM, 75%; (C) IgG: 920 pM, 78%; bisFab: 65 pM, 67%.

Figure 5. Correlation between the potencies of antibodies in the bisFab or full-length IgG formats. EC₅₀ numbers obtained from the fitted dose-response curves for the full-length IgG or bisFab constructs were transformed to their log₁₀ values and analyzed by linear regression.