A PSMA-targeted bispecific antibody for prostate cancer driven by a small-molecule targeting ligand

Sung Chang Lee, Jennifer S Y Ma, Min Soo Kim, Eduardo Laborda, Sei-Hyun Choi, Eric N Hampton, Hwayoung Yun, Vanessa Nunez, Michelle T Muldong, Christina N Wu, Wenxue Ma, Anna A Kulidjian, Christopher J Kane, Vadim Klyushnichenko, Ashley K Woods, Sean B Joseph, Mike Petrassi, John Wisler, Jing Li, Christina A M Jamieson, Peter G Schultz, Chan Hyuk Kim, Travis S Young, Sung Chang Lee, Jennifer S Y Ma, Min Soo Kim, Eduardo Laborda, Sei-Hyun Choi, Eric N Hampton, Hwayoung Yun, Vanessa Nunez, Michelle T Muldong, Christina N Wu, Wenxue Ma, Anna A Kulidjian, Christopher J Kane, Vadim Klyushnichenko, Ashley K Woods, Sean B Joseph, Mike Petrassi, John Wisler, Jing Li, Christina A M Jamieson, Peter G Schultz, Chan Hyuk Kim, Travis S Young

Abstract

Despite the development of next-generation antiandrogens, metastatic castration-resistant prostate cancer (mCRPC) remains incurable. Here, we describe a unique semisynthetic bispecific antibody that uses site-specific unnatural amino acid conjugation to combine the potency of a T cell-recruiting anti-CD3 antibody with the specificity of an imaging ligand (DUPA) for prostate-specific membrane antigen. This format enabled optimization of structure and function to produce a candidate (CCW702) with specific, potent in vitro cytotoxicity and improved stability compared with a bispecific single-chain variable fragment format. In vivo, CCW702 eliminated C4-2 xenografts with as few as three weekly subcutaneous doses and prevented growth of PCSD1 patient-derived xenograft tumors in mice. In cynomolgus monkeys, CCW702 was well tolerated up to 34.1 mg/kg per dose, with near-complete subcutaneous bioavailability and a PK profile supporting testing of a weekly dosing regimen in patients. CCW702 is being evaluated in a first in-human clinical trial for men with mCRPC who had progressed on prior therapies (NCT04077021).

Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).

Figures

Fig. 1. Affinity optimization of DUPA.
Fig. 1. Affinity optimization of DUPA.
(A) General structure of the DUPA–linker–anti-CD3 Fab (DUPA-CD3) conjugate showing the unnatural amino acid p-acetylphenylalanine and oxime ligation (bond) formed between the ketone and amino-oxy group. (B) Chemical structures of DUPA and DUPA with P-Phthal, and P-TriA linkers with measured PSMA inhibition constant (Ki) shown from fig. S1. (C). In vitro cytotoxicity assay by lactate dehydrogenase (LDH) release, of coculture of PBMCs with C4-2 cells [E:T (Effector to Target ratio) = 10:1], 24 hours, comparing dose titration of UCHT1-2× Phthal-DUPA and UCHT1-2× P-TriA-DUPA with wild-type (wt) UCHT1 Fab that was not conjugated to DUPA. UCHT1 is the anti-CD3 antibody clone used in (10). Each data point is a mean of three samples, and error bars represent SD. Sigmoidal dose response (variable slope) curve fit was applied.
Fig. 2. Characterization of deimmunized and optimized…
Fig. 2. Characterization of deimmunized and optimized Fab-DUPA conjugates.
Flow cytometry–based binding curves of deimmunized anti-CD3 Fab (huL5H2_DI)–2× P-TriA-DUPA conjugates to (A) human CD3+ Jurkat cells and (B) cynomolgus monkey CD3+ HSC-F cells. MFI, mean fluorescence intensity. EC50 (nM) by sigmoidal four parameter logistic (4PL) curve fit is tabulated below. (C) In vitro cytotoxicity assay by LDH release of coculture of PBMCs with C4-2 cells (E:T = 10:1), 24 hours, of candidate huL5H2_DI–P-TriA-DUPA by monovalent 1× LC or 1× HC, or bivalent 2× LC/HC. (D) Real-time cytotoxicity assay (XCELLigence) comparing 1× HC (green), 1× LC (red), 2× LC/HC (blue), and pasotuxizumab comparator (purple), each at 100 pM. Full curves at range of concentrations shown in fig. S4.
Fig. 3. Functional comparison of bivalent (2×…
Fig. 3. Functional comparison of bivalent (2× LC/HC) versus monovalent (1× HC) DUPA-CD3 conjugates.
(A) In vitro cytotoxicity by LDH release of coculture of PBMCs with PSMA-positive C4-2 or PSMA-negative DU145 prostate cancer cells (E:T = 10:1), 24 hours. (B) Cytokine release from PBMCs in culture conditions from (A). Supernatants from cocultures were taken after 24 hours and shown for C4-2 for 0.01, 0.1, and 1.0 nM 2× LC/HC or 1× HC conjugates. Little to no cytokines were detectable from coculture with DU145. (C) Up-regulation of T cell activation markers CD25 and CD69 (% double-positive cells) by flow cytometry following coculture conditions in (A). **P ≤ 0.0021 significance by two-way analysis of variance (ANOVA). (D) T cell proliferation after coculture with C4-2 cells and DUPA–anti-CD3 conjugates by carboxyfluorescein succinimidyl ester (CFSE) dilution in flow cytometry after 72 hours (E:T = 1:1). Population doublings are shown above graphs with percentage cells in each doubling listed in inset. In (A) to (D), 2× LC/HC candidates are shown in red and 1× HC in blue.
Fig. 4. Profiling of CCW702 in vitro…
Fig. 4. Profiling of CCW702 in vitro binding and activity.
(A) Octet binding affinities of CCW702 (huL5H2_DI-1× P-TriA-DUPA) to recombinant PSMA and CD3 (δ/ε chains) from mouse, cynomolgus monkey, and human. Hu, human; Cy, cynomolgus; Mu, murine; and KD, dissociation constant. (B) Summary table of in vitro cytotoxicity, cytokine production, and up-regulation of activation markers CD69 and CD25 of PBMCs cocultured with a dose titration of CCW702 in the presence of PSMA-positive C4-2 target cells, average of two normal donor-derived PBMCs. Curve fits by sigmoidal 4PL are shown in fig. S8. (C) Correlation (sigmoidal, 4PL) between surface expression of PSMA in human prostate cancer cell lines (quantified by flow in fig. S9) and in vitro cytotoxicity [shown in (B)]. Parental 22Rv-1 cells exhibited two distinct population on PSMA expression; sorting of 22Rv-1 cells was performed to enrich for PSMA-expressing cells. (D) Effect of soluble PSMA on CCW702-mediated T cell activation via Jurkat-NFAT-luc cells, 20 hours at 37°C. RLU, relative light units. Each data point is a mean of three samples, and error bars represent SD.
Fig. 5. CCW702 in vivo antitumor efficacy…
Fig. 5. CCW702 in vivo antitumor efficacy in xenograft models in immunodeficient NSG mice.
(A and B) CCW702 dose titration in C4-2 xenograft with dosing of CCW702 and pasotuxizumab every other day (QAD) and every 4 days (Q4D) or every 7 days (Q7D). CCW702, pasotuxizumab, and controls were dosed subcutaneously except where indicated as intravenously (iv). CCW702 tumors were injected subcutaneously on day 0; 20 × 106 expanded human T cells were delivered intraperitoneally on day 7, followed by treatment initiation on day 8, n = 7 per group. Spider plots and body weight for each group and individual mouse are shown in figs. S11 and S12. (C) CCW702 antitumor efficacy in a bone metastasis patient-derived xenograft (PDX) model, PCSD1. CCW702 was administered intravenously, 0.2 mg/kg, daily for 10 days starting at days 31 and 60. PCSD1 tumors were injected intrafemorally on day 0; 20 × 106 expanded human T cells were delivered intraperitoneally at day 30, followed by treatment initiation at day 31, n = 10 per group. BLI (Bioluminescence imaging) is shown in fig. S16. For both models, data shown represent mean tumor volume ± SEM. Dotted lines indicate administration of described treatment. Significance, ****P ≤ 0.0001 by two-way ANOVA and Tukey’s multiple comparisons post-test. IF, intrafemoral.
Fig. 6. Pharmacodynamics of CCW702 administered subcutaneously…
Fig. 6. Pharmacodynamics of CCW702 administered subcutaneously to male cynomolgus monkeys.
(A) Heatmaps of cytokines measured from peripheral blood of animals administrated CCW702 at 2, 9.8, and 34.1 μg/kg, every other day for 10 days (a total of five total doses). Time points of collection at the left represent hours after the first dose (day 1) and the fifth dose (day 9), separated by a horizontal dashed line. Log2 color scale: red, 104 pg/ml; black 103 pg/ml; and green, 102 pg/ml. No color (white) indicates that detection was below limit of quantification. (B) Enumeration of CD8 (left) and CD4 (right) T cells by flow cytometry from animals administrated with CCW702 at 2, 9.8, and 34.1 μg/kg, sampled after the first and last dose of CCW702. Black triangles with numbers and vertical dotted lines represent dose.

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