Broadening the Therapeutic Window of ADCs Using Site-Specific Bioconjugation Showcased by an MMAE-Containing Peptide Linker in a CD79b-Targeting ADC

Abstract

The limitations of first-generation antibody-drug conjugate (ADC) technologies include suboptimal stability and efficacy, poor safety profiles, and challenging manufacturing processes. In this study, we describe an anti-CD79b-monomethyl auristatin E (MMAE) ADC generated using a novel peptide-based linker technology that allows for site-specific linker-payload conjugation to native antibodies in only one step. The ADC comprises native polatuzumab as the targeting antibody and a linker-payload consisting of a RKAA-peptide linker and MMAE. We compared our anti-CD79b-RKAA-MMAE ADC with polatuzumab vedotin (PV), the FDA-approved ADC for diffuse large B-cell lymphoma. In the clinic, PV shows significant instability in circulation, leading to strong and dose-limiting side effects, including neutropenia and peripheral neuropathy. The anti-CD79b-RKAA-MMAE ADC showed optimal biophysical properties with a well-defined drug-to-antibody ratio of 2. It demonstrated potent cytotoxicity in multiple cancer cell lines and was very stable in mouse, cynomolgus monkey, and human sera. The anti-CD79b-RKAA-MMAE conjugate showed equal antitumor efficacy at half the payload dose compared with PV in different xenograft models. At equal MMAE concentrations, greater tumor growth inhibition and a considerably longer duration of response were observed. Ultimately, the highest nonseverely toxic dose of 30 mg/kg was determined in a 4-week repeat-dose toxicology study in rats, which is a 3-fold higher ADC dose than reported for PV. In summary, the data show that our novel site-specific bioconjugation technology enabled the generation of an anti-CD79b-RKAA-MMAE ADC with highly favorable biophysical properties and a greatly improved therapeutic index by a factor of 4 to 6 compared with PV. The ADC may therefore represent a safe and efficacious alternative for patients with diffuse large B-cell lymphoma.

Graphical abstract

Figure 1.

Concept of novel one-step and site-specific MTG technology and manufacturing of anti–CD79b-RKAA-MMAE. A, Schematic drawing of the novel MTG-based conjugation technology using a linker-payload composed of a small RKAA peptide linker and the cytotoxic payload MMAE. Functionalization occurs at the Q295 residue of the native, glycosylated polatuzumab antibody. RKAA-MMAE is attached in a single step using wild-type MTG. B, Linker-payload structure comprised the RKAA-peptide linker with an acetylated N-terminus, a PABC self-immolative spacer, and MMAE. C, Schematic comparison of anti–CD79b-RKAA-MMAE generated by one-step site-specific MTG conjugation and FDA-approved PV.

Figure 2.

Analytic characterization of anti–CD79b-RKAA-MMAE. A, LC/MS spectra of the heavy chain (HC) and light chain (LC) of glycosylated anti–CD79b-RKAA-MMAE under reducing conditions. Main peaks showing single conjugation of HC (left graph) and unconjugated LC (right graph). B, Intact LC/MS. Main peaks showing DAR 2 ADC. C, Reverse-phase UHPLC analysis of reduced anti–CD79b-RKAA-MMAE. D, Analysis of monomer content by SEC. E, Comparison of HIC profiles of unconjugated polatuzumab antibody, anti–CD79b-RKAA-MMAE, and PV. Different DAR species of the preparations are indicated.

Figure 3.

In vitro evaluation of binding, toxicity, and stability of anti–CD79b-RKAA-MMAE. A, Binding comparison of unconjugated polatuzumab, anti–CD79b-RKAA-MMAE, and PV on recombinant CD79b by ELISA. The graph shows mean ± SD of technical duplicates. B, Representative assay to evaluate target binding on CD79b-positive BJAB cells. C, Evaluation of Fc functionality by ELISA on immobilized FcγRI (CD64). D,In vitro cytotoxicity assay on CD79b-expressing Granta-519 cells. Comparison of anti–CD79b-RKAA-MMAE (DAR 2) and PV (DAR 3.5) at equal ADC concentrations. E, Toxicity on CD79b-positive WSU-DLCL2 tumor cells. F,In vitro cytotoxicity assay on target-negative HT cells. G, All cytotoxicity graphs show mean ± SD of technical duplicates. A representative serum stability assay of anti–CD79b-RKAA-MMAE in human, cynomolgus monkey, and mouse IgG-depleted sera at 37°C for 7 days. DAR was assessed by LC/MS at different time points. H, Evaluation of PV stability in human, cynomolgus monkey, and mouse IgG-depleted sera. MFI, mean fluorescence intensity; OD, optical density.

Figure 4.

Enzymatic cleavage of anti–CD79b-RKAA-MMAE. Comparison of the cleavage rate of anti–CD79b-RKAA-MMAE and PV using human cathepsin B (A), mouse Ces1c (B), or HLL extract (C). D, Schematic overview of the cleavage mechanism of anti–CD79b-RKAA-MMAE by proteases and subsequent payload release and liberation of free MMAE by 1,6-elimination of PABC.

Figure 5.

Pharmacokinetic studies in Swiss wild-type mice and Sprague Dawley rats. A, Healthy female Swiss wild-type mice were injected intravenously with 5 mg/kg unconjugated polatuzumab, anti–CD79b-RKAA-MMAE, or PV (n = 5 per group). Plasma was collected at different time points after injection, and total IgG and intact ADC concentrations were determined by ELISA. Data represent mean plasma concentrations (±SD). B, Male Sprague Dawley rats were injected intravenously with 10 mg/kg of anti–CD79b-RKAA-MMAE (top graph) or 10 mg/kg of PV (bottom graph) in a repeat-dose study (n = 5 per group). Animals were injected weekly for a total of four injections. Graphs show plasma concentration–time curves after the fourth dose, including the predose concentration measurement (0 hours).

Figure 6.

Therapeutic efficacy of anti–CD79b-RKAA-MMAE in different xenograft models. Treatment was started when tumors reached a size of approximately 200 mm³. Mice were allocated into different groups using a nonrandom stratification protocol. Data represent mean tumor volumes (±SEM). A, CB17-SCID mice were challenged with 20 × 10⁶ Granta-519 tumor cells and received a single injection (black arrow) of 2.1 mg/kg anti–CD79b-RKAA-MMAE (corresponding to 20 μg/kg of payload), 2.1 mg/kg PV (corresponding to 40 μg/kg of payload), or PBS, intravenously into the lateral tail vein (n = 8 per group). B, Treatment of the Granta-519 xenograft model at equal payload doses of 10 µg/kg, corresponding to 1 mg/kg of anti–CD79b-RKAA-MMAE or 0.53 mg/kg of PV (n = 8 per group). C, Administration of 1.5 mg/kg (15 μg/kg of payload) of anti–CD79b-RKAA-MMAE or nonbinding control IgG1-RKAA-MMAE to Granta-519–bearing CB17-SCID mice. D, CB17-SCID mice were challenged with 20 × 10⁶ Ramos Burkitt lymphoma cells. Anti–CD79b-RKAA-MMAE (2.5 mg/kg) or PV (1.43 mg/kg; both corresponding to 25 μg/kg of payload) was used for treatment (n = 6 per group). PBS was used as a negative control. *, P < 0.05; ****, P < 0.0001. CR, complete response.