Predictive biomarkers for sacituzumab govitecan efficacy in Trop-2-expressing triple-negative breast cancer.

Sacituzumab govitecan (SG) is an antibody-drug conjugate composed of a humanized anti-Trop-2 IgG antibody conjugated a hydrolysable linker to SN-38, the topoisomerase I-inhibitory active component of irinotecan. We investigated whether Trop-2-expression and homologous recombination repair (HRR) of SN-38-mediated double-strand DNA (dsDNA) breaks play a role in the sensitivity of triple-negative breast cancer (TNBC) to SG. Activation of HRR pathways, as evidenced by Rad51 expression, was assessed in SG-sensitive cell lines with low and moderate Trop-2-expression (SK-MES-1 squamous cell lung carcinoma and HCC1806 TNBC, respectively), compared to a low Trop-2-expressing, less SG-sensitive TNBC cell line (MDA-MB-231).

IMMU-140, a Novel SN-38 Antibody-Drug Conjugate Targeting HLA-DR, Mediates Dual Cytotoxic Effects in Hematologic Cancers and Malignant Melanoma.

HLA-DR is a member of the MHC class II antigen family expressed on hematologic and solid tumors. Antibodies directed against HLA-DR have demonstrated some clinical success, but toxicities limited development. IMMU-140 is an anti-HLA-DR antibody-drug conjugate composed of the active metabolite of irinotecan, SN-38, conjugated to a humanized anti-HLA-DR IgG antibody (IMMU-114); the IgG naked antibody is devoid of immune functions.

Combination Therapy with Bispecific Antibodies and PD-1 Blockade Enhances the Antitumor Potency of T Cells.

The DOCK-AND-LOCK (DNL) method is a platform technology that combines recombinant engineering and site-specific conjugation to create multispecific, multivalent antibodies of defined composition with retained bioactivity. We have applied DNL to generate a novel class of trivalent bispecific antibodies (bsAb), each comprising an anti-CD3 scFv covalently conjugated to a stabilized dimer of different antitumor Fabs. Here, we report the further characterization of two such constructs, (E1)-3s and (14)-3s, which activate T cells and target Trop-2- and CEACAM5-expressing cancer cells, respectively.

Synthetic Lethality Exploitation by an Anti-Trop-2-SN-38 Antibody-Drug Conjugate, IMMU-132, Plus PARP Inhibitors in -wild-type Triple-Negative Breast Cancer.

Both PARP inhibitors (PARPi) and sacituzumab govitecan (IMMU-132) are currently under clinical evaluation in triple-negative breast cancer (TNBC). We sought to investigate the combined DNA-damaging effects of the topoisomerase I (Topo I)-inhibitory activity of IMMU-132 with PARPi disruption of DNA repair in TNBC., human TNBC cell lines were incubated with IMMU-132 and various PARPi (olaparib, rucaparib, or talazoparib) to determine the effect on growth, double-stranded DNA (dsDNA) breaks, and cell-cycle arrest.

Redirected T-cell killing of solid cancers targeted with an anti-CD3/Trop-2-bispecific antibody is enhanced in combination with interferon-α.

Trop-2 has limited presence on normal tissues but is highly expressed in diverse epithelial cancers. (E1)-3s is a T-cell-redirecting trivalent bispecific antibody (bsAb), comprising an anti-CD3 scFv covalently linked to a stabilized dimer of a Trop-2-targeting Fab using Dock-and-Lock. We show for the first time that bsAb-mediated bidirectional trogocytosis occurs between target and T cells and involves immunologic synapses.

A new class of bispecific antibodies to redirect T cells for cancer immunotherapy.

Various constructs of bispecific antibodies (bsAbs) to redirect effector T cells for the targeted killing of tumor cells have shown considerable promise in both preclinical and clinical studies. The single-chain variable fragment (scFv)-based formats, including bispecific T-cell engager (BiTE) and dual-affinity re-targeting (DART), which provide monovalent binding to both CD3 on T cells and to the target antigen on tumor cells, can exhibit rapid blood clearance and neurological toxicity due to their small size (~55 kDa). Herein, we describe the generation, by the modular DOCK-AND-LOCK™) (DNL™) method, of novel T-cell redirecting bispecific antibodies, each comprising a monovalent anti-CD3 scFv covalently conjugated to a stabilized dimer of different anti-tumor Fabs.

Trogocytosis of multiple B-cell surface markers by CD22 targeting with epratuzumab.

Epratuzumab, a humanized anti-CD22 antibody, is currently in clinical trials of B-cell lymphomas and autoimmune diseases, demonstrating therapeutic activity in non-Hodgkin lymphoma (NHL) and systemic lupus erythematosus (SLE). Thus, epratuzumab offers a promising option for CD22-targeted immunotherapy, yet its mechanism of action remains poorly understood. Here we report for the first time that epratuzumab promptly induces a marked decrease of CD22 (>80%), CD19 (>50%), CD21 (>50%), and CD79b (>30%) on the surface of B cells in peripheral blood mononuclear cells (PBMCs) obtained from normal donors or SLE patients, and of NHL cells (Daudi and Raji) spiked into normal PBMCs.

Evaluation of a novel hexavalent humanized anti-IGF-1R antibody and its bivalent parental IgG in diverse cancer cell lines.

A major mechanism of monoclonal antibodies that selectively target the insulin-like growth factor type 1 receptor (IGF-1R) to inhibit tumor growth is by downregulating the receptor, regardless whether they are capable (antagonistic) or incapable (agonistic) of blocking the binding of cognate ligands. We have developed and characterized a novel agonistic anti-IGF-1R humanized antibody, hR1, and used the Dock-and-Lock (DNL) method to construct Hex-hR1, the first multivalent antibody comprising 6 functional Fabs of hR1, with the aim of enhancing potency of hR1. Based on cross-blocking experiments, hR1 recognizes a region of cysteine-rich domain on the α-subunit, different from the epitopes mapped for existing anti-IGF-1R antibodies, yet hR1 is similar to other anti-IGF-1R antibodies in downregulating IGF-1R and inhibiting proliferation, colony formation, or invasion of selected cancer cell lines in vitro, as well as suppressing growth of the RH-30 rhabdomyosarcoma xenograft in nude mice when combined with the mTOR inhibitor, rapamycin.

Preclinical studies on targeted delivery of multiple IFNα2b to HLA-DR in diverse hematologic cancers.

The short circulating half-life and side effects of IFNα affect its dosing schedule and efficacy. Fusion of IFNα to a tumor-targeting mAb (mAb-IFNα) can enhance potency because of increased tumor localization and improved pharmacokinetics. We used the Dock-and-Lock method to generate C2-2b-2b, a mAb-IFNα comprising tetrameric IFNα2b site-specifically linked to hL243 (humanized anti-HLA-DR).

A new mammalian host cell with enhanced survival enables completely serum-free development of high-level protein production cell lines.

With over 25 monoclonal antibodies (mAbs) currently approved and many more in development, there is considerable interest in gaining improved productivity by increasing cell density and enhancing cell survival of production cell lines. In addition, high costs and growing safety concerns with use of animal products have made the availability of serum-free cell lines more appealing. We elected to transfect the myeloma cell line Sp2/0-Ag14 with Bcl2-EEE, the constitutively active phosphomimetic mutant of Bcl2, for extended cell survival.

A bispecific antibody-IFNalpha2b immunocytokine targeting CD20 and HLA-DR is highly toxic to human lymphoma and multiple myeloma cells.

The short circulating half-life and side effects of IFNα affect its dosing schedule and efficacy. Fusion of IFNα to a tumor-targeting monoclonal antibody (MAb-IFNα) can enhance potency due to increased tumor localization and improved pharmacokinetics. We report the generation and characterization of the first bispecific MAb-IFNα, designated 20-C2-2b, which comprises two copies of IFNα2b and a stabilized F(ab)(2) of hL243 (humanized anti-HLA-DR; IMMU-114) site-specifically linked to veltuzumab (humanized anti-CD20).