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 via 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). Further, two Trop-2-transfectants of MDA-MB-231, C13 and C39 (4- and 25-fold higher Trop-2, respectively), were treated in mice with SG to determine whether increasing Trop-2 expression improves SG efficacy. SG mediated >2-fold increase in Rad51 in MDA-MB-231 but had no effect in SK-MES-1 or HCC1806, resulting in lower levels of dsDNA breaks in MDA-MB-231. SG and saline produced similar effects in parental MDA-MB-231 tumor-bearing mice (median survival time (MST) = 21d and 19.5d, respectively). However, in mice bearing higher Trop-2-expressing C13 and C39 tumors after Trop-2 transfection, SG provided a significant survival benefit, even compared to irinotecan (MST = 97d vs. 35d for C13, and 81d vs. 28d for C39, respectively; P < 0.0007). These results suggest that SG could provide better clinical benefit than irinotecan in patients with HRR-proficient tumors expressing high levels of Trop-2, as well as to patients with HRR-deficient tumors expressing low/moderate levels of Trop-2.

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 IgG4 antibody (IMMU-114); the IgG4 naked antibody is devoid of immune functions. Our aim was to determine if SN-38, the metabolite of a drug not commonly used in hematopoietic cancers, would be effective and safe when targeted to HLA-DR-expressing tumors. IMMU-140 had dual-therapeutic mechanisms, as evidenced by its retention of nonoverlapping anti-HLA-DR nonclassical apoptotic signaling and classical apoptosis mediated by its SN-38 payload. In seven human disease models [acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), multiple myeloma (MM), acute myeloid leukemia (AML), diffuse large B-cell lymphoma (DLBCL), Hodgkin lymphoma (HL), and melanoma], IMMU-140 provided significant therapeutic efficacy compared with controls, in vitro, in 3D spheroid models, and in vivo Except for MM and HL, IMMU-140 imparted significantly improved antitumor effects compared with parental IMMU-114. Even in intractable AML and ALL, where IMMU-114 only had modest antitumor effects, IMMU-140 therapy mediated >80% improvement in survival. Therapy was well tolerated, as demonstrated by no marked loss in body weight. Combined with doxorubicin, IMMU-140 produced significantly greater antitumor effects in HL than with monotherapy and without any added toxicity. The dual-therapeutic action of IMMU-140 resulted in promising therapeutic activity in a range of hematopoietic tumors and melanoma, and therefore warrants clinical development. Mol Cancer Ther; 17(1); 150-60. ©2017 AACR.

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. (E1)-3s and (14)-3s, in the presence of human T cells, killed target cells grown as monolayers at subnanomolar concentrations, with a similar potency observed for drug-resistant cells. Antitumor efficacy was demonstrated for (E1)-3s coadministered with human peripheral blood mononuclear cells (PBMC) in NOD/SCID mice harboring xenografts of MDA-MB-231, a triple-negative breast cancer line constitutively expressing Trop-2 and PD-L1. Growth inhibition was observed following treatment with (E1)-3s or (14)-3s combined with human PBMC in 3D spheroids generated from target cell lines to mimic the in vivo behavior and microenvironment of these tumors. Moreover, addition of an antagonistic anti-PD-1 antibody increased cell death in 3D spheroids and extended survival of MDA-MB-231-bearing mice. These preclinical results emphasize the potential of combining T-cell-redirecting bsAbs with antagonists or agonists that mitigate T-cell inhibition within the tumor microenvironment to improve immunotherapy of solid cancers in patients. They also support the use of 3D spheroids as a predictive alternative to in vivo models for evaluating T-cell functions. Cancer Res; 77(19); 5384-94. ©2017 AACR.

Synthetic Lethality Exploitation by an Anti-Trop-2-SN-38 Antibody-Drug Conjugate, IMMU-132, Plus PARP Inhibitors in BRCA1/2-wild-type Triple-Negative Breast Cancer.

Purpose: 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.Experimental Design:In vitro, 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. Mice bearing BRCA1/2-mutated or -wild-type human TNBC tumor xenografts were treated with the combination of IMMU-132 and PARPi (olaparib or talazoparib). Study survival endpoint was tumor progression to >1.0 cm3 and tolerability assessed by hematologic changes.Results: Combining IMMU-132 in TNBC with all three different PARPi results in synergistic growth inhibition, increased dsDNA breaks, and accumulation of cells in the S-phase of the cell cycle, regardless of BRCA1/2 status. A combination of IMMU-132 plus olaparib or talazoparib produces significantly improved antitumor effects and delay in time-to-tumor progression compared with monotherapy in mice bearing BRCA1/2-mutated HCC1806 TNBC tumors. Furthermore, in mice bearing BRCA1/2-wild-type tumors (MDA-MB-468 or MDA-MB-231), the combination of IMMU-132 plus olaparib imparts a significant antitumor effect and survival benefit above that achieved with monotherapy. Most importantly, this combination was well tolerated, with no substantial changes in hematologic parameters.Conclusions: These data demonstrate the added benefit of combining Topo I inhibition mediated by IMMU-132 with synthetic lethality provided by PARPi in TNBC, regardless of BRCA1/2 status, thus supporting the rationale for such a combination clinically. Clin Cancer Res; 23(13); 3405-15. ©2017 AACR.

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. We studied the effects of interferon-α (INFα) on (E1)-3s-mediated T-cell killing of human gastric and pancreatic cancer cell lines. T-cell activation, cytokine induction, and cytotoxicity were evaluated ex vivo using peripheral blood mononuclear cells (PBMC) or T cells with NCI-N87 gastric cancer as target cells. In vivo activity was assayed with NCI-N87 and Capan-1 (pancreatic) xenografts. In the presence of target cells and PBMCs, (E1)-3s did not cause excess cytokine production. When combined with (E1)-3s, peginterferonalfa-2a--which alone did not increase T-cell activation or raise cytokine levels over baseline--increased CD69 expression but did not significantly increase cytokine induction. (E1) 3s mediated a highly potent T-cell lysis of NCI-N87 target cells in vitro. Inclusion of peginterferonalfa-2a or a more potent form of INFα, 20*-2b, significantly potentiated the activity of (E1)-3s by more than 2.5- or 7-fold, respectively. In vivo, combining peginterferonalfa-2a with (E1)-3s delayed Capan-1 growth longer than each single agent. Similarly, combination therapy delayed tumor proliferation of NCI-N87 compared with (E1)-3s or peginterferonalfa-2a single-treatment groups. (E1)-3s effectively induced T-cell-mediated killing of Trop-2-expressing pancreatic and gastric cancers, which was enhanced with INFα.

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. The potential advantages of this design include bivalent binding to tumor cells, a larger size (~130 kDa) to preclude renal clearance and penetration of the blood-brain barrier, and potent T-cell mediated cytotoxicity. These prototypes were purified to near homogeneity, and representative constructs were shown to provoke the formation of immunological synapses between T cells and their target tumor cells in vitro, resulting in T-cell activation and proliferation, as well as potent T-cell mediated anti-tumor activity. In addition, in vivo studies in NOD/SCID mice bearing Raji Burkitt lymphoma or Capan-1 pancreatic carcinoma indicated statistically significant inhibition of tumor growth compared with untreated controls.

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. Although some Fc-independent loss of CD22 is expected from internalization by epratuzumab, the concurrent and prominent reduction of CD19, CD21, and CD79b is Fc dependent and results from their transfer from epratuzumab-opsonized B cells to FcγR-expressing monocytes, natural killer cells, and granulocytes via trogocytosis. The findings of reduced levels of CD19 are implicative for the efficacy of epratuzumab in autoimmune diseases because elevated CD19 has been correlated with susceptibility to SLE in animal models as well as in patients. This was confirmed herein by the finding that SLE patients receiving epratuzumab immunotherapy had significantly reduced CD19 compared with treatment-naïve patients.

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. Hex-hR1 and hR1 are generally comparable in their bioactivities under the in-intro and in-vivo conditions investigated. Nevertheless, in selective experiments involving a direct comparison of potency, Hex-hR1 demonstrated a stronger effect on inhibiting cell proliferation stimulated by IGF-1 and could effectively downregulate IGF-1R at a concentration as low as 20 pM.

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). In vitro, C2-2b-2b inhibited various B-cell lymphoma leukemia and myeloma cell lines. In most cases, this immunocytokine was more effective than CD20-targeted mAb-IFNα or a mixture comprising the parental mAb and IFNα. Our findings indicate that responsiveness depends on HLA-DR expression/density and sensitivity to IFNα and hL243. C2-2b-2b induced more potent and longer-lasting IFNα signaling compared with nontargeted IFNα. Phosphorylation of STAT1 was more robust and persistent than that of STAT3, which may promote apoptosis. C2-2b-2b efficiently depleted lymphoma and myeloma cells from whole human blood but also exhibited some toxicity to B cells, monocytes, and dendritic cells. C2-2b-2b showed superior efficacy compared with nontargeting mAb-IFNα, peginterferonalfa-2a, or a combination of hL243 and IFNα, using human lymphoma and myeloma xenografts. These results suggest that C2-2b-2b should be useful in the treatment of various hematopoietic malignancies.

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. After adaptation of the initial transfectants to serum-independent growth, a clone with superior growth properties, referred to as SpESF, was isolated and further subjected to iterative rounds of stressful growth over a period of 4 months. The effort resulted in the selection of a promising clone, designated SpESFX-10, which was shown to exhibit robust growth and resist apoptosis induced by sodium butyrate or glutamine deprivation. The advantage of SpESFX-10 as a host for generating mAb-production cell lines was demonstrated by its increased transfection efficiency, culture longevity, and mAb productivity, as well as by the feasibility of accomplishing the entire cell line development process, including transfection, subcloning, and cryopreservation, in the complete absence of serum.

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). In vitro, 20-C2-2b inhibited each of four lymphoma and eight myeloma cell lines, and was more effective than monospecific CD20-targeted MAb-IFNα or a mixture comprising the parental antibodies and IFNα in all but one (HLA-DR(-)/CD20(-)) myeloma line, suggesting that 20-C2-2b should be useful in the treatment of various hematopoietic malignancies. 20-C2-2b displayed greater cytotoxicity against KMS12-BM (CD20(+)/HLA-DR(+) myeloma) compared with monospecific MAb-IFNα, which targets only HLA-DR or CD20, indicating that all three components in 20-C2-2b could contribute to toxicity. Our findings indicate that a given cell's responsiveness to MAb-IFNα depends on its sensitivity to IFNα and the specific antibodies, as well as the expression and density of the targeted antigens.