Cross-arm binding efficiency of an EGFR x c-Met bispecific antibody

Figures & data

Figure 1. (A) Schematic representation of the cross-arm binding efficiency (χ), (B) the ligand-binding model structure for JNJ-61186372, an EGFR x c-Met bispecific antibody.

Figure 2. Simultaneous fitting of the EGFR and c-Met monovalent parent (gp120 x EGFR Ab and gp120 x c-Met Ab) binding data to 8 NSCLC cell lines, model fitting (line) vs. observed data (symbols).

Table 1. EGFR and c-Met receptor densities and model derived cell densities of the 8 NSCLC cell lines used in flow cytometry staining.

Cell line	c-Met receptor #	EGF receptor #	Model estimated cell density
H1993	5.61 × 10^5	3.25 × 10^5	5.43 × 10^6
SNU-5	4.93 × 10^5	1.11 × 10^5	4.94 × 10^6
H3255	1.34 × 10^5	8.23 × 10^5	1.59 × 10^7
H1975	8.61 × 10^4	6.32 × 10^4	1.79 × 10^7
H292	6.12 × 10^4	3.57 × 10^5	5.73 × 10^6
HCC4006	5.69 × 10^4	9.09 × 10^4	1.96 × 10^7
H1650	5.68 × 10^4	1.21 × 10^5	7.53 × 10^6
SKMES-1	4.60 × 10^4	1.67 × 10^5	7.89 × 10^6

Table 2. Model input parameter and model-estimated parameter values.

Parameters	Estimate	Relative standard error%
kON1 (1/nM/min) for EGFR monovalent binding	0.0276	N/A
kOFF1 (1/min) for EGFR monovalent binding	0.166	3.80%
VMEDIA (L)	0.000175	N/A
kON2 (1/nM/min) for c-Met monovalent binding	0.0398	N/A
kOFF2 (1/min) for c-Met monovalent binding	0.0028	N/A
EGFR baseline conc. (nM) H3255	10.8	6.90%
EGFR baseline conc. (nM) HCC4006	0.909	7.50%
EGFR baseline conc. (nM) H292	1.99	6.30%
EGFR baseline conc. (nM) H1993	1.75	9%
EGFR baseline conc. (nM) SKMES-1	1.31	6.10%
EGFR baseline conc. (nM) SNU-5	0.558	8.70%
EGFR baseline conc. (nM) H1650	0.729	4.80%
EGFR baseline conc. (nM) H1975	1.12	10.40%
c-Met baseline conc. (nM) for H3255	1.918	N/A
c-Met baseline conc. (nM) for HCC4006	1.113	N/A
c-Met baseline conc. (nM) H292	0.348	N/A
c-Met baseline conc. (nM) H1993	2.908	N/A
c-Met baseline conc. (nM) SKMES-1	0.352	N/A
c-Met baseline conc. (nM) SNU-5	2.363	N/A
c-Met baseline conc. (nM) H1650	0.422	N/A
c-Met baseline conc. (nM) H1975	1.544	N/A
cross-arm binding efficiency (χ)	104	37%

* Fixed values in the “Step 1: Determination of the monovalent binding affinities of JNJ-61186372 to EGFR and c-Met on cell surfaces” estimation process based on ProteOn data.

# For each cell line, the “c-Met baseline receptor concentration” is specified as “EGFR baseline receptor conc. × the ratio between c-Met and EGFR receptor numbers per cell” in the differential equations. Values shown in table for c-Met baseline receptor concentrations were computed based on model estimated EGFR baseline receptor concentrations.

^• Parameter estimate based on “Step 2: Determination of χ for JNJ-61186372”.

Figure 3. Estimation of the χ value of JNJ-61186372 by simultaneously fitting the ligand-binding model to JNJ-61186372 binding data to 3 NSCLC cell lines with similar EGFR and c-Met receptor densities.

Figure 4. (A) Model-predicted binding of JNJ-61186372 to EGFR and c-Met, respectively, on H1993 and H292; (B) Correlation between the model-predicted EC₅₀ for EGFR and c-Met binding and the observed IC₅₀ values for EGFR and c-Met phosphorylation inhibition in H1993 and H292 cell lines.

Figure 5. Simulated profiles of the free fraction of EGFR and c-Met in the H1975/HGF mouse xenograft model for JNJ-61186372 and the combination of both monovalent parent antibodies (gp120 x EGFR Ab + B21M x c-Met Ab).

Figure 6. Tumor growth inhibition profiles by JNJ-61186372 (10mg/kg) and the combination therapy of both monovalent antibodies (10mg/kg+10mg/kg) in a H1975/HGF mouse xenograft model (Dosing = BIW × 3 weeks) *P < 0.05, ^#P < 0.1 based on a 2-tailed t-test for each studied day.

Figure 7. Model simulation to show the effect of χ on the binding of a bispecific antibody (BsAb) to receptors (A) χ has more significant effect on the binding of a BsAb to the second receptor (R2), than the anchor receptor (R1, which is assumed to have 10-fold higher abundance and 10-fold higher affinity); (B) The effect of χ on R2 binding is more apparent when the R2 baseline density is lower (R1 is assumed to have 10-fold higher affinity).