Sacituzumab Govitecan (IMMU-132), an Anti-Trop-2/SN-38 Antibody-Drug Conjugate: Characterization and Efficacy in Pancreatic, Gastric, and Other Cancers.

Sacituzumab govitecan (IMMU-132) is an antibody-drug conjugate (ADC) made from a humanized anti-Trop-2 monoclonal antibody (hRS7) conjugated with the active metabolite of irinotecan, SN-38. In addition to its further characterization, as the clinical utility of IMMU-132 expands to an ever-widening range of Trop-2-expressing solid tumor types, its efficacy in new disease models needs to be explored in a nonclinical setting. Unlike most ADCs that use ultratoxic drugs and stable linkers, IMMU-132 uses a moderately toxic drug with a moderately stable carbonate bond between SN-38 and the linker. Flow cytometry and immunohistochemistry disclosed that Trop-2 is expressed in a wide range of tumor types, including gastric, pancreatic, triple-negative breast (TNBC), colonic, prostate, and lung. While cell-binding experiments reveal no significant differences between IMMU-132 and parental hRS7 antibody, surface plasmon resonance analysis using a Trop-2 CM5 chip shows a significant binding advantage for IMMU-132 over hRS7. The conjugate retained binding to the neonatal receptor, but it lost greater than 60% of the antibody-dependent cell-mediated cytotoxicity activity compared to that of hRS7. Exposure of tumor cells to either free SN-38 or IMMU-132 demonstrated the same signaling pathways, with pJNK1/2 and p21(WAF1/Cip1) upregulation followed by cleavage of caspases 9, 7, and 3, ultimately leading to poly-ADP-ribose polymerase cleavage and double-stranded DNA breaks. Pharmacokinetics of the intact ADC in mice reveals a mean residence time (MRT) of 15.4 h, while the carrier hRS7 antibody cleared at a similar rate as that of the unconjugated antibody (MRT ∼ 300 h). IMMU-132 treatment of mice bearing human gastric cancer xenografts (17.5 mg/kg; twice weekly × 4 weeks) resulted in significant antitumor effects compared to that of mice treated with a nonspecific control. Clinically relevant dosing schemes of IMMU-132 administered either every other week, weekly, or twice weekly in mice bearing human pancreatic or gastric cancer xenografts demonstrate similar, significant antitumor effects in both models. Current Phase I/II clinical trials ( ClinicalTrials.gov , NCT01631552) confirm anticancer activity of IMMU-132 in cancers expressing Trop-2, including gastric and pancreatic cancer patients.

Improving the therapeutic index in cancer therapy by using antibody-drug conjugates designed with a moderately cytotoxic drug.

The antibody-drug conjugate (ADC), IMMU-130, of the moderately cytotoxic topoisomerase I inhibitor, SN-38, and the CEACAM5-targeted humanized antibody (mAb), labetuzumab, was evaluated in model systems of human colon carcinoma and in phase I clinical trials of heavily pretreated patients with metastatic colorectal cancer. The conjugate, designed with a near-homogeneous drug substitution of 7-8 SN-38/mAb and with a linker that released 50% of the drug in ∼20 h, showed significant antitumor effects compared to a nontargeted ADC in human tumor xenografts, which could be augmented in combination with bevacizumab. The advantage of fractionated dosing was demonstrated, with potential implications for the clinical dosing schedule. Biodistribution comparing IMMU-130 with labetuzumab showed that the conjugate cleared somewhat faster from the blood, but this did not affect tumor uptake and retention. The use of an ultrastable linker in the conjugate design abrogated antitumor effects. A tolerability study in rabbits showed a high safety margin, with no-observed-adverse-effect level (NOAEL) corresponding to a cumulative human-equivalent protein dose of 40-60 mg/kg. The preclinical findings appear to be corroborated in two phase I clinical trials, with high tolerability and evidence of antitumor activity, including objective responses. The impact of the ADC design on the utility of IMMU-130, tailored to a poorly internalizing target, is discussed.

Trop-2-targeting tetrakis-ranpirnase has potent antitumor activity against triple-negative breast cancer.

BACKGROUND: Ranpirnase (Rap) is an amphibian ribonuclease with reported antitumor activity, minimal toxicity, and negligible immunogenicity in clinical studies, but the unfavorable pharmacokinetics and suboptimal efficacy hampered its further clinical development. To improve the potential of Rap-based therapeutics, we have used the DOCK-AND-LOCK™ (DNL™) method to construct a class of novel IgG-Rap immunoRNases. In the present study, a pair of these constructs, (Rap)2-E1-(Rap)2 and (Rap)2-E1*-(Rap)2, comprising four copies of Rap linked to the CH3 and CK termini of hRS7 (humanized anti-Trop-2), respectively, were evaluated as potential therapeutics for triple-negative breast cancer (TNBC). METHODS: The DNL-based immunoRNases, (Rap)2-E1-(Rap)2 and (Rap)2-E1*-(Rap)2, were characterized and tested for biological activities in vitro on a panel of breast cancer cell lines and in vivo in a MDA-MB-468 xenograft model. RESULTS: (Rap)2-E1-(Rap)2 was highly purified (>95%), exhibited specific cell binding and rapid internalization in MDA-MB-468, a Trop-2-expressing TNBC line, and displayed potent in vitro cytotoxicity (EC50 ≤ 1 nM) against diverse breast cancer cell lines with moderate to high expression of Trop-2, including MDA-MB-468, BT-20, HCC1806, SKBR-3, and MCF-7. In comparison, structural counterparts of (Rap)2-E1-(Rap)2, generated by substituting hRS7 with selective non-Trop-2-binding antibodies, such as epratuzumab (anti-CD22), were at least 50-fold less potent than (Rap)2-E1-(Rap)2 in MDA-MB-468 and BT-20 cells, both lacking the expression of the cognate antigen. Moreover, (Rap)2-E1-(Rap)2 was less effective (EC50 > 50 nM) in MDA-MB-231 (low Trop-2) or HCC1395 (no Trop-2), and did not show any toxicity to human peripheral blood mononuclear cells. In a mouse TNBC model, a significant survival benefit was achieved with (Rap)2-E1*-(Rap)2 when given the maximal tolerated dose. CONCLUSIONS: A new class of immunoRNases was generated with enhanced potency for targeted therapy of cancer. The promising results from (Rap)2-E1-(Rap)2 and (Rap)2-E1*-(Rap)2 support their further investigation as a potential treatment option for TNBC and other Trop-2-expressing cancers.

Dual-targeting immunotherapy of lymphoma: potent cytotoxicity of anti-CD20/CD74 bispecific antibodies in mantle cell and other lymphomas.

We describe the use of novel bispecific hexavalent Abs (HexAbs) to enhance anticancer immunotherapy. Two bispecific HexAbs [IgG-(Fab)(4) constructed from veltuzumab (anti-CD20 IgG) and milatuzumab (anti-CD74 IgG)] show enhanced cytotoxicity in mantle cell lymphoma (MCL) and other lymphoma/leukemia cell lines, as well as patient tumor samples, without a crosslinking Ab, compared with their parental mAb counterparts, alone or in combination. The bispecific HexAbs have different properties from and are more potent than their parental mAbs in vitro. The juxtaposition of CD20 and CD74 on MCL cells by the HexAbs resulted in homotypic adhesion and triggered intracellular changes that include loss of mitochondrial transmembrane potential, production of reactive oxygen species, rapid and sustained phosphorylation of ERKs and JNK, down-regulation of pAkt and Bcl-xL, actin reorganization, and lysosomal membrane permeabilization, culminating in cell death. They also displayed different potencies in depleting lymphoma cells and normal B cells from whole blood ex vivo and significantly extended the survival of nude mice bearing MCL xenografts in a dose-dependent manner, thus indicating stability and antitumor activity in vivo. Such bispecific HexAbs may constitute a new class of therapeutic agents for improved cancer immunotherapy, as shown here for MCL and other CD20(+)/CD74(+) malignancies.

Improving Intracellular Delivery of an Antibody-Drug Conjugate Targeting Carcinoembryonic Antigen Increases Efficacy at Clinically Relevant Doses In Vivo.

Solid tumor antibody-drug conjugates (ADC) have experienced more clinical success in the last 5 years than the previous 18-year span since the first ADC approval in 2000. While recent advances in protein engineering, linker design, and payload variations have played a role in this success, high expression and readily internalized targets have also been crucial to solid tumor therapy. However, these factors are also paradoxically connected to poor tissue penetration and lower efficacy. Previous work shows that potent ADCs can benefit from slower internalization under subsaturating doses to improve tissue penetration and increase tumor response. In contrast, faster internalization is predicted to increase efficacy under higher, tumor saturating doses. In this work, the intracellular delivery of SN-38 conjugated to an anti-carcinoembryonic antigen (anti-CEA) antibody (Ab) is increased by coadministering a noncompeting (cross-linking) anti-CEA Ab to improve efficacy in a colorectal carcinoma animal model. The SN-38 payload enables broad tumor saturation with clinically-tolerable doses, and under these saturating conditions, using a second CEA receptor cross-linking Ab yields faster internalization, which increases tumor killing efficacy. Our spheroid results show indirect bystander killing can also occur, but the more efficient direct cell killing from targeted intracellular payload release drives a greater tumor response. These results provide a strategy to increase therapeutic effectiveness with improved intracellular delivery under tumor saturating doses with the potential to expand the ADC target repertoire.

Sacituzumab govitecan plus platinum-based chemotherapy mediates significant antitumor effects in triple-negative breast, urinary bladder, and small-cell lung carcinomas.

Sacituzumab govitecan (SG) is an antibody-drug conjugate composed of an anti-Trop-2-directed antibody conjugated with the topoisomerase I inhibitory drug, SN-38, via a proprietary hydrolysable linker. SG has received United States Food and Drug Administration (FDA) approval to treat metastatic triple-negative breast cancer (TNBC), unresectable locally advanced or metastatic hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative breast cancer, and accelerated approval for metastatic urothelial cancer. We investigated the utility of combining SG with platinum-based chemotherapeutics in TNBC, urinary bladder carcinoma (UBC), and small-cell lung carcinoma (SCLC). SG plus carboplatin or cisplatin produced additive growth-inhibitory effects in vitro that trended towards synergy. Immunoblot analysis of cell lysates suggests perturbation of the cell-cycle and a shift towards pro-apoptotic signaling evidenced by an increased Bax to Bcl-2 ratio and down-regulation of two anti-apoptotic proteins, Mcl-1 and survivin. Significant antitumor effects were observed with SG plus carboplatin in mice bearing TNBC or SCLC tumors compared to all controls (P < 0.0062 and P < 0.0017, respectively) and with SG plus cisplatin in UBC and SCLC tumor-bearing animals (P < 0.0362 and P < 0.0001, respectively). These combinations were well tolerated by the animals. Combining SG with platinum-based chemotherapeutics demonstrates the benefit in these indications and warrants further clinical investigation.

Prevention of acute graft-versus-host disease in a xenogeneic SCID mouse model by the humanized anti-CD74 antagonistic antibody milatuzumab.

Prevention and treatment of graft-versus-host disease (GVHD) remains a major challenge, given that current T-cell depletion and mainstay immunosuppressive therapies compromise preexisting T-cell immunity, often leading to severe infections and disease relapse. Thus, there is a critical need for novel anti-GVHD agents that can spare protective T-cell memory. Here we show that milatuzumab (hLL1), a humanized anti-CD74 antagonist monoclonal antibody, can moderately reduce the numbers of CD74-expressing B cells and myeloid dendritic cells, but has no effect on the survival of T cells that are CD74(-). Consequently, milatuzumab inhibits allogeneic T-cell proliferation in mixed leukocyte reactions. In a human/mouse xenogeneic SCID mouse model in which GVHD is induced and mediated by engrafted human CD4(+) T cells and dendritic cells, milatuzumab effectively prevents the onset and manifestations of acute GVHD, suppresses serum levels of human IFN-γ and IL-5, eliminates the infiltration of human lymphocytes into GVHD target organs (ie, lung, liver, and spleen), and significantly promotes survival (90% versus 20% for controls; P = .0012). Importantly, exposure to milatuzumab does not affect the number of cytomegalovirus-specific, IFN-γ-producing human CD8(+) T cells in allogeneic mixed leukocyte reactions. These encouraging results warrant further exploration of milatuzumab as a possible new therapeutic agent for GVHD.

Optimization of multivalent bispecific antibodies and immunocytokines with improved in vivo properties.

Multifunctional antibody-based biologics, such as bispecific antibodies and immunocytokines, can be difficult to produce with sufficient yield and stability, and often exhibit inferior pharmacokinetics. Dock-and-Lock (DNL) is a modular method that combines recombinant engineering with site-specific conjugation, allowing the construction of various complex, yet defined, biostructures with multivalency and multispecificity. The technology platform exploits the natural interaction between two interactive human protein binding domains that are modified to provide covalent fusion. We explored the potential application of a new class of IgG-based DNL modules with an anchor domain fused at the C-terminal end of the kappa light chain (C(k)), instead of the C-terminal end of the Fc. Two C(k)-derived prototypes, an anti-CD22/CD20 bispecific hexavalent antibody, comprising epratuzumab (anti-CD22) and four Fabs of veltuzumab (anti-CD20), and a CD20-targeting immunocytokine, comprising veltuzumab and four molecules of interferon-α2b, were compared to their Fc-derived counterparts. The Ck-based conjugates exhibited superior Fc-effector functions in vitro, as well as improved pharmacokinetics, stability, and anti-lymphoma activity in vivo. These results favor the selection of DNL conjugates with the C(k)-design for future clinical development.

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.

Targeting both IGF-1R and mTOR synergistically inhibits growth of renal cell carcinoma in vitro.

BACKGROUND: Advanced or metastatic renal cell carcinoma (RCC) has a poor prognosis, because it is relatively resistant to conventional chemotherapy or radiotherapy. Treatments with human interferon-α2b alone or in combination with mammalian target of rapamycin (mTOR) inhibitors have led to only a modest improvement in clinical outcome. One observation made with mTOR inhibitors is that carcinomas can overcome these inhibitory effects by activating the insulin-like growth factor-I (IGF-I) signaling pathway. Clinically, there is an association of IGF-I receptor (IGF-IR) expression in RCC and poor long-term patient survival. We have developed a humanized anti-IGF-IR monoclonal antibody, hR1, which binds to RCC, resulting in effective down-regulation of IGF-IR and moderate inhibition of cell proliferation in vitro. In this work, we evaluate the anti-tumor activity of two novel IGF-1R-targeting agents against renal cell carcinoma given alone or in combination with an mTOR inhibitor. METHODS: hR1 was linked by the DOCK-AND-LOCK™ (DNL™) method to four Fabs of hR1, generating Hex-hR1, or to four molecules of interferon-α2b, generating 1R-2b. Eight human RCC cell lines were screened for IGF-1R expression and sensitivity to treatment with hR1 in vitro. Synergy with an mTOR inhibitor, temsirolimus, was tested in a cell line (ACHN) with low sensitivity to hR1. RESULTS: Hex-hR1 induced the down-regulation of IGF-IR at 10-fold lower concentrations compared to the parental hR1. Sensitivity to growth inhibition mediated by hR1 and Hex-hR1 treatments correlated with IGF-1R expression (higher expression was more sensitive). The potency of 1R-2b to inhibit the in vitro growth of RCC was also demonstrated in two human cell lines, ACHN and 786-O, with EC50-values of 63 and 48 pM, respectively. When combined with temsirolimus, a synergistic growth-inhibition with hR1, Hex-hR1, and 1R-2b was observed in ACHN cells at concentrations as low as 10 nM for hR1, 1 nM for Hex-hR1, and 2.6 nM for 1R-2b. CONCLUSIONS: Both Hex-hR1 and 1R-2b proved to be more potent than parental hR1 in inhibiting growth of RCC in vitro. Synergy was achieved when each of the three hR1-based agents was combined with temsirolimus, suggesting a new approach for treating RCC.

Antibody-Drug Conjugate Sacituzumab Govitecan Drives Efficient Tissue Penetration and Rapid Intracellular Drug Release.

Antibody-drug conjugates (ADC) are a rapidly growing class of targeted cancer treatments, but the field has experienced significant challenges from their complex design. This study examined the multiscale distribution of sacituzumab govitecan (SG; Trodelvy), a recently clinically approved ADC, to clarify the mechanism(s) of efficacy given its unique design strategy. We employed a multiscale quantitative pharmacokinetic approach, including near-infrared fluorescence imaging, single-cell flow cytometry measurements, payload distribution via γH2AX pharmacodynamic staining, and a novel dual-labeled fluorescent technique to track the ADC and payload in a high trophoblast cell-surface antigen 2 expression xenograft model of gastric cancer (NCI-N87). We found that rapid release of the SN-38 payload from the hydrolysable linker inside cells imparts more DNA damage in vitro and in vivo than an ADC with a more stable enzyme cleavable linker. With SG, little to no extracellular payload release in the tumor was observed using a dual-labeled fluorescence technique, although bystander effects were detected. The high dosing regimen allowed the clinical dose to reach the majority of cancer cells, which has been linked to improved efficacy. In addition, the impact of multiple doses (day 1 and day 8) of a 21-day cycle was found to further improve tissue penetration despite not changing tumor uptake [percent injected dose per gram (%ID/g)] of the ADC. These results show increased ADC efficacy with SG can be attributed to efficient tumor penetration and intracellular linker cleavage after ADC internalization. This quantitative approach to study multiscale delivery can be used to inform the design of next-generation ADCs and prodrugs for other targets.

Therapy of B-cell malignancies by anti-HLA-DR humanized monoclonal antibody, IMMU-114, is mediated through hyperactivation of ERK and JNK MAP kinase signaling pathways.

A humanized IgG4 anti-HLA-DR monoclonal antibody (IMMU-114), engineered to avoid side effects associated with complement activation, was examined for binding and cytotoxicity on leukemia, lymphoma, and multiple myeloma cell lines and chronic lymphocytic leukemia (CLL) patient specimens, followed by evaluation of the effects of IMMU-114 on extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling pathways. HLA-DR was expressed on the majority of these cells at markedly higher levels than CD20, CD22, and CD74. IMMU-114 was toxic to mantle cell lymphoma, CLL, acute lymphoblastic leukemia, hairy cell leukemia, non-Hodgkin lymphoma (including rituximab-resistant), and multiple myeloma cell lines, and also patient CLL cells. IMMU-114 induced disease-free survival in tumor-bearing SCID mice with early-stage disease and in models that are relatively resistant to anti-CD20 monoclonal antibodies. Despite positive staining, acute myelogenous leukemic cells were not killed by IMMU-114. The ability of IMMU-114 to induce activation of ERK and JNK signaling correlated with cytotoxicity and differentiates the mechanism of action of IMMU-114 from monoclonal antibodies against CD20 and CD74. Thus, antigen expression is not sufficient for cytotoxicity; antibody-induced hyperactivation of ERK and JNK mitogen activated protein kinase signaling pathways are also required.

Signal amplification in molecular imaging by pretargeting a multivalent, bispecific antibody.

Here we describe molecular imaging of cancer using signal amplification of a radiotracer in situ by pretargeting a multivalent, bispecific antibody to carcinoembryonic antigen (CEA), which subsequently also captures a radioactive hapten-peptide. Human colon cancer xenografts as small as approximately 0.15 g were disclosed in nude mice within 1 h of giving the radiotracer, with tumor/blood ratios increased by >or=40-fold (approximately 10:1 at 1 h, approximately 100:1 at 24 h), compared to a (99m)Tc-labeled CEA-specific F(ab') used clinically for colorectal cancer detection, while also increasing tumor uptake tenfold ( approximately 20% injected dose/g) under optimal conditions. This technology could be adapted to other antibodies and imaging modalities.

Hexavalent bispecific antibodies represent a new class of anticancer therapeutics: 1. Properties of anti-CD20/CD22 antibodies in lymphoma.

The dock and lock (DNL) method is a new technology for generating multivalent antibodies. Here, we report in vitro and in vivo characterizations of 20-22 and 22-20, a pair of humanized hexavalent anti-CD20/22 bispecific antibodies (bsAbs) derived from veltuzumab (v-mab) and epratuzumab (e-mab). The 22-20 was made by site-specific conjugation of e-mab to 4 Fabs of v-mab; 20-22 is of the opposite configuration, composing v-mab and 4 Fabs of e-mab. Each bsAb translocates both CD22 and CD20 into lipid rafts, induces apoptosis and growth inhibition without second-antibody crosslinking, and is significantly more potent in killing lymphoma cells in vitro than their parental antibodies. Although both bsAbs triggered antibody-dependent cellular toxicity, neither displayed complement-dependent cytotoxicity. Intriguingly, 22-20 and 20-22 killed human lymphoma cells in preference to normal B cells ex vivo, whereas the parental v-mab depleted malignant and normal B cells equally. In vivo studies in Daudi tumors revealed 20-22, despite having a shorter serum half-life, had antitumor efficacy comparable with equimolar v-mab; 22-20 was less potent than 20-22 but more effective than e-mab and control bsAbs. These results indicate multiple advantages of hexavalent anti-CD20/22 bsAbs over the individual parental antibodies and suggest that these may represent a new class of cancer therapeutics.

CD20-targeted tetrameric interferon-alpha, a novel and potent immunocytokine for the therapy of B-cell lymphomas.

Interferon-alpha (IFN-alpha) has direct inhibitory effects on some tumors and is a potent stimulator of both the innate and adaptive immune systems. A tumor-targeting antibody-IFN-alpha conjugate (mAb-IFN-alpha) could kill by direct actions of the monoclonal antibody (mAb) and IFN-alpha on tumor cells and also potentiate a tumor-directed immune response. The modular Dock-and-Lock method (DNL) was used to generate 20-2b, the first immunocytokine having 4 cytokine (IFN-alpha2b) groups that are fused to the humanized anti-CD20 mAb, veltuzumab. Additional mAb-IFN-alpha constructs, each retaining potent IFN-alpha2b biologic activity, also were produced by DNL. The 20-2b shows enhanced antibody-dependent cellular cytotoxicity compared with veltuzumab but lacks complement-dependent cytotoxicity. The 20-2b inhibits in vitro proliferation of lymphoma cells and depletes them from whole human blood more potently than the combination of veltuzumab and a nontargeting, irrelevant, mAb-IFN-alpha. The 20-2b demonstrated superior therapeutic efficacy compared with veltuzumab or nontargeting mAb-IFN-alpha in 3 human lymphoma xenograft models, even though mouse immune cells respond poorly to human IFN-alpha2b. Targeting IFN-alpha with an anti-CD20 mAb makes the immunocytokine more potent than either agent alone. These findings suggest that 20-2b merits clinical evaluation as a new candidate antilymphoma therapeutic.

Properties and structure-function relationships of veltuzumab (hA20), a humanized anti-CD20 monoclonal antibody.

Veltuzumab is a humanized anti-CD20 monoclonal antibody with complementarity-determining regions (CDRs) identical to rituximab, except for one residue at the 101st position (Kabat numbering) in CDR3 of the variable heavy chain (V(H)), having aspartic acid (Asp) instead of asparagine (Asn), with framework regions of epratuzumab, a humanized anti-CD22 antibody. When compared with rituximab, veltuzumab has significantly reduced off-rates in 3 human lymphoma cell lines tested, as well as increased complement-dependent cytotoxicity in 1 of 3 cell lines, but no other in vitro differences. Mutation studies confirmed that the differentiation of the off-rate between veltuzumab and rituximab is related to the single amino acid change in CDR3-V(H). Studies of intraperitoneal and subcutaneous doses in mouse models of human lymphoma and in normal cynomolgus monkeys disclosed that low doses of veltuzumab control tumor growth or deplete circulating or sessile B cells. Low- and high-dose veltuzumab were significantly more effective in vivo than rituximab in 3 lymphoma models. These findings are consistent with activity in patients with non-Hodgkin lymphoma given low intravenous or subcutaneous doses of veltuzumab. Thus, changing Asn(101) to Asp(101) in CDR3-V(H) of rituximab is responsible for veltuzumab's lower off-rate and apparent improved potency in preclinical models that could translate into advantages in patients.

Regulation of CEACAM5 and Therapeutic Efficacy of an Anti-CEACAM5-SN38 Antibody-drug Conjugate in Neuroendocrine Prostate Cancer.

PURPOSE: Neuroendocrine prostate cancer (NEPC) is an aggressive form of castration-resistant prostate cancer (CRPC) for which effective therapies are lacking. We previously identified carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) as a promising NEPC cell surface antigen. Here we investigated the scope of CEACAM5 expression in end-stage prostate cancer, the basis for CEACAM5 enrichment in NEPC, and the therapeutic potential of the CEACAM5 antibody-drug conjugate labetuzumab govitecan in prostate cancer. EXPERIMENTAL DESIGN: The expression of CEACAM5 and other clinically relevant antigens was characterized by multiplex immunofluorescence of a tissue microarray comprising metastatic tumors from 34 lethal metastatic CRPC (mCRPC) cases. A genetically defined neuroendocrine transdifferentiation assay of prostate cancer was developed to evaluate mechanisms of CEACAM5 regulation in NEPC. The specificity and efficacy of labetuzumab govitecan was determined in CEACAM5+ prostate cancer cell lines and patient-derived xenografts models. RESULTS: CEACAM5 expression was enriched in NEPC compared with other mCRPC subtypes and minimally overlapped with prostate-specific membrane antigen, prostate stem cell antigen, and trophoblast cell surface antigen 2 expression. We focused on a correlation between the expression of the pioneer transcription factor ASCL1 and CEACAM5 to determine that ASCL1 can drive neuroendocrine reprogramming of prostate cancer which is associated with increased chromatin accessibility of the CEACAM5 core promoter and CEACAM5 expression. Labetuzumab govitecan induced DNA damage in CEACAM5+ prostate cancer cell lines and marked antitumor responses in CEACAM5+ CRPC xenograft models including chemotherapy-resistant NEPC. CONCLUSIONS: Our findings provide insights into the scope and regulation of CEACAM5 expression in prostate cancer and strong support for clinical studies of labetuzumab govitecan for NEPC.

Correction: Trop-2 is a novel target for solid cancer therapy with sacituzumab govitecan (IMMU-132), an antibody-drug conjugate (ADC).

[This corrects the article DOI: 10.18632/oncotarget.4318.].

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.

A new method to produce monoPEGylated dimeric cytokines shown with human interferon-α2b.

We have adapted the dock-and-lock (DNL) method into a novel PEGylation technology using human interferon-α2b (IFN-α2b) as an example. Central to DNL is a pair of distinct protein domains involved in the natural association between cAMP-dependent protein kinase (PKA) and A-kinase anchoring proteins (AKAPs). These domains serve as linkers for site-specific conjugation of poly(ethylene glycol) (PEG) to a dimeric form of IFN-α2b. The combination of a fusion protein comprising IFN-α2b and the dimerization-and-docking domain (DDD) of PKA with a PEG-derivatized anchoring domain (AD) of an interactive AKAP results in facile formation of a trimeric complex containing two copies of IFN-α2b and a single site-specifically linked PEG chain. Three such monoPEGylated dimers of IFN-α2b have been generated, the first with a 20 kDa linear PEG, referred to as α2b-362, the second with a 30 kDa linear PEG (α2b-413), and the third with a 40 kDa branched PEG (α2b-457). All three retained antiviral and antitumor activity in vitro and showed improved pharmacokinetic properties in mice, which translated into potent and prolonged therapeutic efficacy in the Daudi human lymphoma xenograft model. We anticipate wide applicability of the DNL method for developing long-acting therapeutics that are dimeric and monoPEGylated with the increased bioavailability allowing for less frequent dosing.