Anti-BCMA/CD16A Bispecific Molecule (RO7297089)

The anti-BCMA/CD16A molecule was in-licensed from Affimed GmbH, and RO7297089 was generated by Genentech. It is comprised of a humanized anti-BCMA antibody with two identical light and heavy chains of human IgG1 isotype and two anti-CD16A single-chain variable fragments (scFv) that are genetically fused to the C-terminal end of the IgG heavy chain residue G446 (EU nomenclature) via a (G4S)6 linker sequence. Additional amino acid changes were made to impair binding to human Fc gamma-receptors and attenuate Fc-mediated effector function.

Animal Housing and Procedure

The single-dose cynomolgus monkey study was performed at the Covance Preclinical Services GmbH test facility (Germany) and the repeat-dose cynomolgus monkey study was performed at the Covance Laboratories (UK). Both sites were fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. All study procedures complied with the Animal Welfare Act and were approved by the local Institutional Animal Care and Use Committee.

Single-Dose Cynomolgus Monkey Study

A total of eight healthy male cynomolgus monkeys (2.9–7.0 kg) and four healthy female cynomolgus monkeys (2.9–4.5 kg) were included in the study. All animals were between 2 and 3 years old. Two males and one female were randomly assigned to each treatment group and received a single 1-h intravenous (IV) infusion of vehicle solution, or 5, 15, or 45 mg/kg of RO7297089. Approximately 1 mL of blood was sampled from the vena femoralis of each individual animal at multiple time points post-drug administration (Supplementary Fig. 1A). Blood samples were processed to obtain serum and evenly aliquoted for measuring unbound RO7297089 and ADA. All animals were euthanized via sedation by intramuscular injection of ketamine hydrochloride, followed by an IV sodium pentobarbital overdose prior to exsanguination. All collected samples were stored at −70°C before assessment.

Repeat-Dose Cynomolgus Monkey Study

The repeat-dose study was conducted in healthy cynomolgus monkeys between 2 and 3 years old (2.5–3.1 kg). Two male and two female animals were randomly assigned to each treatment group and received weekly 2-h IV infusion of vehicle solution, or 15, or 50 mg/kg/injection of RO7297089 for five injections. Approximately 0.2–0.5 mL blood was sampled at previously specified time points (Supplementary Fig. 1B) to isolate serum for assessing total RO7297089 concentration and ADA. To measure total sBCMA and sCD16 concentration in systemic circulation, 1.0 mL blood was obtained from the femoral or cephalic vein of each animal at pre-dose and 3, 7, 14, 21, 28, and 30 days post-treatment initiation and split into two tubes to measure target levels. Blood was centrifuged to obtain plasma. In addition to total sBCMA and sCD16 levels, systemic IgM level and RNA expression of plasma cells markers (such as J-chain and BCMA) (18) were evaluated and presented previously (10). All animals were euthanized using the same method described above. All samples were stored at −60 to −80°C before assessment.

Assay Development

Unbound RO7297089 concentration was measured by an ELISA assay with CD16A-human Fc for capture, BCMA-rabbit Fc for detection, and a horseradish peroxidase (HRP) conjugated donkey anti-rabbit IgG as secondary detection. The lower limit of quantitation (LLOQ) of the assay was 0.39 nM in cynomolgus monkey sera.

Total RO7297089 concentration (including unbound RO7297089, sBCMA-RO7297089 complex, RO7297089-sCD16 complex, and sBCMA-RO7297089-sCD16 complex) was analyzed using a method entitled a hybrid immunoaffinity capture liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for the quantitation of RO7297089 in cynomolgus monkey serum, which was validated at Pharmaceutical Product Development (PPD), LLC. The LLOQ for neat samples was 25.0 nM. The method and validation of the assay for the measurement of RO7207089 is similar to what is described previously (19).

Total sBCMA concentration (including unbound sBCMA, sBCMA-RO7297089 complex, and sBCMA-RO7297089-sCD16 complex) was assessed using a hybrid immunoaffinity capture LC-MS/MS assay. The assay uses biotinylated goat anti-BCMA polyclonal antibody (does not compete with RO7297089 for BCMA binding) for capture. Streptavidin magnetic beads were then used to enrich the immune-complex. Because BCMA is too large for practical direct quantitative analysis using LC-MS/MS technology, the analyte captured on the beads was subjected to proteolysis with trypsin, following standard protein denaturation, reduction, and alkylation processing steps. The characteristic peptide fragments produced by this procedure can then be quantified as surrogates of soluble BCMA concentrations by LC-MS/MS (i.e., multiple reaction monitoring or MRM). The LLOQ for neat samples was 5 nM.

Total soluble CD16 concentration (including unbound sCD16, RO7297089-sCD16 complex, and sBCMA-RO7297089-sCD16 complex) was assessed using an ELISA. Plasma samples diluted 1/10 in buffer containing 75 μg/mL of RO7297089 were incubated before being added to a 96-well streptavidin microplate containing biotinylated anti-CD16a rabbit monoclonal antibody (does not compete with RO7297089 for CD16 binding). Human BCMA-Fc conjugated to digoxigenin was then added for detection. Horseradish peroxidase (HRP)-conjugated mouse monoclonal anti-digoxin followed by KPL TMB microwell peroxidase substrate were added for color development. The reaction was stopped with 1.0 M phosphoric acid and the plate was read on a platereader at 450 nm. The data was analyzed with a 4 parameter fit and 1/Y weighting factor. The LLOQ for neat samples was 2.5 nM.

The detection of ADAs to RO7297089 was performed using a bridging ELISA that utilized biotin-conjugated RO7297089 and digoxigenin (DIG)-conjugated RO7297089 to capture ADAs and an HRP-conjugated mouse anti-DIG antibody for detection. Using a mouse anti-human IgG antibody as a surrogate positive control, the relative sensitivity was determined to be 48.2 ng/mL, and the assay was able to detect 1000 ng/mL of the surrogate positive control in the presence of up to 400 ug/mL of RO7297089.

Data Analyses and PK Parameter Calculation

All graphs were plotted using Prism (GraphPad Inc., CA). Nominal sample collection times and nominal dose solution concentrations were used in data analysis. All PK analyses were based on individual animal data. Data from both ADA-positive and -negative animals were included in the PK parameter calculation. The PK parameters from both single-dose and repeat-dose cynomolgus monkey studies were calculated using non-compartmental analyses with Phoenix® WinNonlin® version 6.4 (Certara USA, Inc., NJ). The following PK parameters were calculated for the single-dose cynomolgus monkey study: maximum observed concentration (Cmax); area under the serum concentration-time curve from the end of infusion to the last measurable time point (AUC0-last); area under the concentration-time curve from day 0 to infinity (AUC0-inf); percentage of the AUC0-inf area determined by extrapolation (AUCinf %Extra); systemic clearance (CL); volume of distribution at steady state (Vss). The following parameters were obtained for the repeat-dose study: maximum observed concentration after first drug administration (Cmax Day 0) and fourth drug administration (Cmax Day 21); area under the serum concentration-time curve in the first dosing interval (AUC0-7) and fourth dosing interval (AUC21-28); area under the serum concentration-time curve from the end of infusion to the last measurable time point (AUC0-last); and accumulation ratio (AR) calculated as AUC21-28 / AUC0-7.

TMDD Model Development

A TMDD model was established to characterize the unbound and total RO7297089, total sBCMA, and total sCD16 concentrations from each individual animal (20, 21). The model structure is presented in the figures and a detailed explanation of the model parameters is shown in the table. The equations that describe the model are shown below:

$$\frac\;}=\frac\cdot A_-\frac\cdot A_-\frac\cdot A_-k_\cdot A_\cdot C_+\left(k_\cdot k_\right)\cdot C_\cdot V_1-k_\cdot A_\cdot C_+\left(k_\cdot k_\right)\cdot C_\cdot V_1-k_\cdot A_\cdot C_+\left(k_\cdot k_\right)\cdot C_\cdot V_1-k_\cdot A_\cdot C_+\left(k_\cdot k_\right)\cdot C_\cdot V_1$$

(1)

$$\frac_\ }=\frac\cdot _-\frac\cdot _$$

(2)

Systemic PK of RO7297089 was described using a two-compartment model (Eqs. 1 and 2). V1 and V2 are volumes of distribution from central and peripheral compartments, respectively. CLD and CL are the intercompartmental and systemic clearance. RO7297089 could bind to both membrane and soluble targets reversibly in the central compartment.

$$\frac_}=_-_\ sBCMA}\cdot _-_\cdot_\cdot \left(_+_+_\right)+\left(_\cdot _\right)\cdot \left(_+_+_\right)$$

(3)

$$\frac_}=_-_\ mBCMA}\cdot _-_-_\cdot _\cdot \left(_+_+_\right)+\left(_\cdot _\right)\cdot \left(_+_+_\right)$$

(4)

$$\frac_}=__B}\cdot _\cdot _+\left(__C}\cdot _\right)\cdot \left(_+_\right)-\left(_\cdot _\left)\cdot _-\right(_\cdot \frac\right)\cdot _-_\cdot _\cdot \left(_+_\right)$$

(5)

$$\frac_}=_\cdot _\cdot _+\left(_\cdot _\right)\cdot \left(_+_\right)-\left(_\cdot _\right)\cdot _-_\cdot _\ \cdot \left(_+_\right)-_\ mBCMA}\cdot _$$

(6)

$$\frac_}=_-_\ sCD16}\cdot _-_\cdot _\cdot \left(_+_+_\right)+\left(_\cdot _\right)\cdot \left(_+_+_\right)$$

(7)

$$\frac_}=_-_\ mCD16}\cdot _-_-_\cdot _\cdot \left(_+_+_\right)+\left(_\cdot _\right)\cdot \left(_+_+_\right)$$

(8)

$$\frac_}=_\cdot _\cdot _+\left(_\cdot _\right)\cdot \left(_+_\right)-\left(_\cdot _\left)\cdot _-\right(_\cdot \frac\right)\cdot _-_\cdot _\cdot \left(_+_\right)$$

(9)

$$\frac_}=_\cdot _\cdot _+\left(_\cdot _\right)\cdot \left(_+_\right)-\left(_\cdot _\right)\cdot _-_\cdot _\cdot \left(_+_\right)-_\ mCD16}\cdot _$$

(10)

$$\frac_}=_\cdot _\cdot _+_\cdot _\cdot _-_\cdot \left(_\cdot _+_\cdot _+_\cdot \frac+_\cdot \frac\right)$$

(11)

$$\frac_}=_\cdot _\cdot _+_\cdot _\cdot _-_\cdot \left(_\cdot _+_\cdot _+_\ mCD16}\right)$$

(12)

$$\frac_}=_\cdot _\cdot _+_\cdot _\cdot _-_\cdot \left(_\cdot _+_\cdot _+_\ mBCMA}\right)$$

(13)

$$\frac_}=_\cdot _\cdot _+_\cdot _\cdot _-_\cdot\left(_\cdot _+_\cdot _+_\ mBCMA}+_\ mCD16}\right)$$

(14)

$$_\ sBCMA}=\frac}\ sBCMA}$$

(15)

$$_\ mBCMA}=\frac}\ mBCMA}$$

(16)

$$_\ sCD16}=\frac}\ sCD16}$$

(17)

$$_\ mCD16}=\frac}\ mCD16}$$

(18)

$$_=_=\frac}\ sBCMA}\cdot _\ (0)$$

(19)

$$_=_+\frac}\ mBCMA}\cdot _\ (0)$$

(20)

$$_=_=\frac}\ sCD16}\cdot _\ (0)$$

(21)

$$_=_+\frac}\ mCD16}\cdot _\ (0)$$

(22)

$$_\ (0)= dose\ \left( nmol/ kg\right)$$

(23)

$$Fraction\ of\ Bound\ mBCMA=\left(1-\frac}\ (0)}\right)\times 100\%$$

(28)

$$Fraction\ of\ Bound\ mCD16=\left(1-\frac}\ (0)}\right)\times 100\%$$

(29)

Target binding kinetics were described using the dissociation constant kD and binding constant kon. Baseline levels of mBCMA, sBCMA, mCD16, and sCD16 are represented as CmBCMA (0), CsBCMA (0), CmCD16 (0), and CsCD16 (0). The kinetics of membrane and soluble targets were described in the model by a first-order elimination and a zero-order synthesis (Eqs. 3–14). The elimination of mBCMA and mCD16 was described as a combination of both target-shedding and endogenous degradation/internalization (Eqs. 4 and 8). The degradation rate constants of mBCMA (kdeg mBCMA), sBCMA (kdeg sBCMA), mCD16 (kdeg mCD16), and sCD16 (kdeg sCD16) were described by 0.693/half-life (Eqs. 15–18). The synthesis rates of both soluble and membrane targets were assumed to be constant during the study since we did not observe significant change of plasma and NK cells during the nonclinical study. Since the shedding of mBCMA or mCD16 was regarded as the only source of sBCMA or sCD16, the shedding rate of mBCMA (kshed_B) or mCD16 (kshed_C) is the synthesis rate of sBCMA or sCD16 (Eqs. 21 and 22). The synthesis rate of mBCMA and mCD16 was expressed as Eqs. 20 and 22. The elimination rates of soluble complexes (i.e., mAb-sBCMA, mAb-sCD16, and mAb-sBCMA-sCD16) were described by the adjusted clearance of RO7297089 (i.e., \(_\bullet \frac\)). The elimination rates of membrane complex (i.e., mAb-mBCMA, mAb-mCD16, mAb-mBCMA-sCD16, mAb-sBCMA-mCD16, and mAb-mBCMA-mCD16) were mainly driven by the membrane target internalization rates. The model parameters were simultaneously estimated using the particle swarm global fitting algorithm and combined error model from MATLAB® (Version 2018b) SimBiology® (MathWorks, MA).

Human Translation with TMDD Model

Several parameters of the TMDD model were adjusted accordingly for human translation. The PK parameters (CLD, CL, V1, and V2) in humans were adjusted using an allometric scaling method described by Deng R, et al. (22). Baseline sBCMA levels in different MM patient populations have been reported by multiple groups and are significantly higher than the levels in healthy cynomolgus monkeys (23,24,25,26). Since RO7297089 is designed for the treatment of RRMM, we used the reported sBCMA level in RRMM patients (26), which was 41-fold higher than the observed sBCMA level in healthy cynomolgus monkeys. mBCMA level in RRMM patients is unknown and therefore needs to be estimated. Since shedding of mBCMA is the only source of sBCMA and it was found positively correlated with the cell surface mBCMA expression and number of malignant plasma cells (24), the model assumed that mBCMA was also 41-fold higher in MM patients compared to healthy cynomolgus monkeys (the same fold-increase with sBCMA level). To address the uncertainty around this assumption, an additional ±30% variation was applied to the mBCMA level estimate. Baseline sCD16A levels in RRMM patients were measured and reported in this manuscript. The mCD16A levels were estimated by the same approach as the estimate of mBCMA, assuming sCD16A and mCD16A have the same fold-change in MM patients compared to cynomolgus monkeys with a ±30% variation included.