Donglin Liu

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.

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

BackgroundRanpirnase (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).MethodsThe 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.ConclusionsA 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.

Horizontal Transmission of Malignancy: In-Vivo Fusion of Human Lymphomas with Hamster Stroma Produces Tumors Retaining Human Genes and Lymphoid Pathology

We report the in-vivo fusion of two Hodgkin lymphomas with golden hamster cheek pouch cells, resulting in serially-transplanted (over 5–6 years) GW-532 and GW-584 heterosynkaryon tumor cells displaying both human and hamster DNA (by FISH), lymphoma-like morphology, aggressive metastasis, and retention of 7 human genes (CD74, CXCR4, CD19, CD20, CD71, CD79b, and VIM) out of 24 tested by PCR. The prevalence of B-cell restricted genes (CD19, CD20, and CD79b) suggests that this uniform population may be the clonal initiating (malignant) cells of Hodgkin lymphoma, despite their not showing translation to their respective proteins by immunohistochemical analysis. This is believed to be the first report of in-vivo cell-cell fusion of human lymphoma and rodent host cells, and may be a method to disclose genes regulating both organoid and metastasis signatures, suggesting that the horizontal transfer of tumor DNA to adjacent stromal cells may be implicated in tumor heterogeneity and progression. The B-cell gene signature of the hybrid xenografts suggests that Hodgkin lymphoma, or its initiating cells, is a B-cell malignancy.

In-Vivo Fusion of Human Cancer and Hamster Stromal Cells Permanently Transduces and Transcribes Human DNA

After demonstrating, with karyotyping, polymerase chain reaction (PCR) and fluorescence in-situ hybridization, the retention of certain human chromosomes and genes following the spontaneous fusion of human tumor and hamster cells in-vivo, it was postulated that cell fusion causes the horizontal transmission of malignancy and donor genes. Here, we analyzed gene expression profiles of 3 different hybrid tumors first generated in the hamster cheek pouch after human tumor grafting, and then propagated in hamsters and in cell cultures for years: two Hodgkin lymphomas (GW-532, GW-584) and a glioblastoma multiforme (GB-749). Based on the criteria of MAS 5.0 detection P-values ≤0.065 and at least a 2-fold greater signal expression value than a hamster melanoma control, we identified 3,759 probe sets (ranging from 1,040 to 1,303 in each transplant) from formalin-fixed, paraffin-embedded sections of the 3 hybrid tumors, which unambiguously mapped to 3,107 unique Entrez Gene IDs, representative of all human chromosomes; however, by karyology, one of the hybrid tumors (GB-749) had a total of 15 human chromosomes in its cells. Among the genes mapped, 39 probe sets, representing 33 unique Entrez Gene IDs, complied with the detection criteria in all hybrid tumor samples. Five of these 33 genes encode transcription factors that are known to regulate cell growth and differentiation; five encode cell adhesion- and transmigration-associated proteins that participate in oncogenesis and/or metastasis and invasion; and additional genes encode proteins involved in signaling pathways, regulation of apoptosis, DNA repair, and multidrug resistance. These findings were corroborated by PCR and reverse transcription PCR, showing the presence of human alphoid (α)-satellite DNA and the F11R transcripts in additional tumor transplant generations. We posit that in-vivo fusion discloses genes implicated in tumor progression, and gene families coding for the organoid phenotype. Thus, cancer cells can transduce adjacent stromal cells, with the resulting progeny having permanently transcribed genes with malignant and other gene functions of the donor DNA. Using heterospecific in-vivo cell fusion, genes encoding oncogenic and organogenic traits may be identified.

Combining ABCG2 Inhibitors with IMMU-132, an Anti–Trop-2 Antibody Conjugate of SN-38, Overcomes Resistance to SN-38 in Breast and Gastric Cancers

Sacituzumab govitecan (IMMU-132), an SN-38–conjugated antibody–drug conjugate, is showing promising therapeutic results in a phase I/II trial of patients with advanced Trop-2–expressing, metastatic, solid cancers. As members of the ATP-binding cassette (ABC) transporters confer chemotherapy resistance by active drug efflux, which is a frequent cause of treatment failure, we explored the use of known inhibitors of ABC transporters for improving the therapeutic efficacy of IMMU-132 by overcoming SN-38 resistance. Two human tumor cell lines made resistant to SN-38, MDA-MB-231-S120 (human breast cancer) and NCI-N87-S120 (human gastric cancer), were established by continuous exposure of the parental cells to stepwise increased concentrations of SN-38 and analyzed by flow cytometry for functional activities of ABCG2 and ABCB1, immunoblotting and qRT-PCR for the expression of ABCG2 at both protein and mRNA levels, and MTS assays for the potency of SN-38 alone or in combination with a modulator of ABC transporters. MDA-MB-231-S120 and NCI-N87-S120 displayed reduced sensitivity to SN-38 in vitro, with IC50 values approximately 50-fold higher than parental MDA-MB-231 and NCI-N87 cells. The increase in drug resistance of both S120 cell populations is associated with the expression of functional ABCG2, but not ABCB1. Importantly, treatment of both S120 sublines with known ABCG2 inhibitors (fumitremorgin C, Ko143, and YHO-13351) restored toxicity of SN-38, and the combination of YHO-13351 with IMMU-132 increased the median survival of mice bearing NCI-N87-S120 xenografts. These results provide a rationale for combination therapy of IMMU-132 and inhibitors of ABC transporters, such as YHO-13351. Mol Cancer Ther; 15(8); 1910–9. ©2016 AACR.

Interferon-λ1 Linked to a Stabilized Dimer of Fab Potently Enhances both Antitumor and Antiviral Activities in Targeted Cells

The type III interferons (IFNs), comprising IFN-λ1, IFN-λ2, and IFN-λ3, behave similarly to IFN-α in eliciting antiviral, antitumor, and immune-modulating activities. Due to their more restricted cellular targets, IFN-λs are attractive as potential alternatives to existing therapeutic regimens based on IFN-αs. We have applied the DOCK-AND-LOCK™ method to improve the anti-proliferative potency of IFN-λ1 up to 1,000-fold in targeted cancer cell lines by tethering stabilized Fab dimers, derived from hRS7 (humanized anti-Trop-2), hMN-15 (humanized anti-CEACAM6), hL243 (humanized anti-HLA-DR), and c225 (chimeric anti-EGFR), to IFN-λ1 site-specifically, resulting in novel immunocytokines designated (E1)-λ1, (15)-λ1, (C2)-λ1, and (c225)-λ1, respectively. Targeted delivery of IFN-λ1 via (15)-λ1 or (c225)-λ1 to respective antigen-expressing cells also significantly increased antiviral activity when compared with non-targeting (C2)-λ1, as demonstrated in human lung adenocarcinoma cell line A549 by (15)-λ1 against encephalomyocarditis virus (EC50 = 22.2 pM versus 223 pM), and in human hepatocarcinoma cell line Huh-7 by (c225)-λ1 against hepatitis C virus (EC50 = 0.56 pM versus 91.2 pM). These promising results, which are attributed to better localization and stronger binding of IFN-λ1 to antibody-targeted cells, together with the favorable pharmacokinetic profile of (E1)-λ1 in mice (T1/2 = 8.6 h), support further investigation of selective prototypes as potential antiviral and antitumor therapeutic agents.

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.

Abstract 3193: Treatment of high Trop-2-expressing triple-negative breast cancer (TNBC) with sacituzumab govitecan (IMMU-132) overcomes homologous recombination repair (HRR) rescue mediated by Rad51

Purpose: IMMU-132 is an antibody-drug conjugate composed of a humanized anti-Trop-2 IgG conjugated via a cleavable linker to SN-38, a topoisomerase I inhibitor and active component of irinotecan. It is currently under clinical investigation in a range of solid tumors (NCT01631552). We investigated the hypothesis that IMMU-132, through its targeting of Trop-2 in solid tumors, will be superior to irinotecan in overcoming Rad51-mediated HRR repair of DNA breaks in TNBC tumors with high Trop-2 expression. Methods: Rad51 and DNA-breaks (γ-H2A.X) were determined by Western blot. Cells with different Trop-2 levels were exposed to IMMU-132 for 24 h (25 - 100 nM SN-38 equivalents), including squamous cell lung carcinoma (SK-MES-1; ~30,000 Trop-2/cell) and TNBC (HCC1806, ~90,000 Trop-2/cell and MDA-MB-231, ~30,000 Trop-2/cell). Also, two Trop-2-transfectants of MDA-MB-231, designated C13 and C39 (4- and 25-fold higher Trop-2 levels, respectively), were likewise exposed to IMMU-132. Mice bearing MDA-MB-231, C13, or C39 tumors were treated with irinotecan (MTD, 40 mg/kg; q2dx5) or IMMU-132 (0.5 mg; 9 μg SN-38 equivalent, twice wkly x 4). Tumors were measured and mice weighed twice weekly. Study survival endpoint was tumor progression to >1.0 cm3. Results: SK-MES-1 and HCC1806 are sensitive to IMMU-132 therapy whereas MDA-MB-231 is resistant. IMMU-132 mediated a >2-fold increase in Rad51 levels in MDA-MB-231 cells, but had no effect in SK-MES-1 or HCC1806. At 25 nM IMMU-132, there were lower levels of DNA breaks detected in MDA-MB-231 relative to SK-MES-1 and HCC1806 (2-fold increase in MDA-MB-231 vs. >3-fold). At higher concentrations of IMMU-132 (100 nM), all 3 cell lines demonstrated similar levels of DNA breaks (~5-fold above background), suggesting that higher levels of SN-38 can overcome Rad51-mediated repair. Both the C13 and C39 clones had a similar response as parental MDA-MB-231 upon IMMU-132 exposure. Mice bearing MDA-MB-231, C13, or C39 tumors treated with irinotecan demonstrated significant improvements in median survival times (MST) compared to saline (P 70d vs. 35d, respectively for C13 and >70d vs. 28d for C39; P Conclusion: IMMU-132, with its unique SN-38-delivery platform, has the potential to provide clinical benefit both to chemo-sensitive solid tumors with low Trop-2 expression, as well as to chemo-resistant tumors with high Trop-2 expression. Citation Format: Thomas M. Cardillo, Ali A. Mostafa, Diane L. Rossi, Donglin Liu, Chien-Hsing Chang, Robert M. Sharkey, David M. Goldenberg. Treatment of high Trop-2-expressing triple-negative breast cancer (TNBC) with sacituzumab govitecan (IMMU-132) overcomes homologous recombination repair (HRR) rescue mediated by Rad51 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3193. doi:10.1158/1538-7445.AM2017-3193

Abstract 1630: Cell-cell fusion in vivo transfers genes for human malignancy, metastasis, and the donor tumors’ organoid phenotype to stromal hamster cells.

Cell-cell fusion in vitro has been a method for investigating oncogenesis, gene mapping, and gene regulation. Whether such mechanisms occur in vivo requires similar studies with in-vivo cell fusion, particularly whether genes for malignancy, metastasis, and organoid features can be transmitted horizontally to adjacent stromal cells. We describe our experiences with the transplantation of a human glioblastoma multiforme and two Hodgkin lymphomas from patient specimens directly to the hamster cheek pouch, resulting in highly malignant tumors that metastasize in their rodent hosts within the initial transplantation passage and subsequently over passage for many years. The resulting glioblastoma cell line, GB-749, showed heterosynkaryon formation between the tumor and hamster host cells by karyology and FISH, and the retention of 7 human genes, VIM, CD74, CXCR4, TP53, PLAGL2, GFAP, and EGFR, by PCR, of which 3 showed protein translation in vivo by immunohistochemistry; namely, CD74, CXCR4, and PLAGL2. These 3 genes have been implicated with malignancy or, particularly, glioblastoma. In the second example of two Hodgkin lymphomas grafted to hamsters, FISH again demonstrated stable human-hamster heterosynkaryons, while PCR showed the retention of 7 human genes in the GW-532 and GW-584 cell lines propagated for 5-6 years in-vivo (VIM, CD74, CXCR4, CD19, CD20, CD71, and CD79b). Thus, the glioblastoma and Hodgkin tumor hybrids retained different genes after cell-cell fusion with hamster cells, but VIM, CD74, and CXCR4 were common, surprisingly, to all three tumor hybrid lines. The hybrid transplants also retained the morphological appearance of their original donor tumors, thus retaining their organoid genes providing the tumor9s morphological phenotype. In the glioblastoma, GB-749, these included PLAGL2, GFAP, and EGFR, while in the Hodgkin lymphoma lines, GW-532 and GW-584, the B-cell-lineage genes, CD19, CD20, CD79b, were retained. We believe these are the first examples of in-vivo cell-cell fusion between human tumor and rodent host cells showing stable heterosynkaryon formation and propagation over many years, and with the retention of select human genes implicated in the malignant phenoytype, metastasis, and organoid gene signatures. Thus, at least in this model, human cancer cells can transmit malignant and organoid genes to stromal cells in their microenvironment, perhaps representing a basic mechanism for the tumor heterogeneity observed clinically. Whether such fusion events occur during the progression of cancers in patients remains to be determined. Citation Format: David M. Goldenberg, Donglin Liu, Meiyu Loo, David V. Gold, Chien-Hsing Chang, Elaine S. Jaffe. Cell-cell fusion in vivo transfers genes for human malignancy, metastasis, and the donor tumors’ organoid phenotype to stromal hamster cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1630. doi:10.1158/1538-7445.AM2013-1630

Abstract 1221: Targeting interferon-λ1 with a stabilized dimer of anti-tumor Fab .

Background: The type-III interferons (IFNs), comprising IFN-λ1, IFN-λ2, and IFN-λ3, are functionally similar to the type-I IFN-α in eliciting anti-viral, anti-tumor, and immune-modulating activities. Due to its more restricted cellular targets, IFN-λ is being considered as a potential alternative to existing therapeutic regimens based on IFN-α, whose adverse effects in patients limit its use. The development of antibody-targeted IFN-λ may further improve its safety, potency and pharmacokinetics. Methods: We generated three DOC-AND-LOCKTM (DNLTM) complexes, designated (E1)-λ1, (15)-λ1, and (C2)-λ1, by conjugating IFN-λ1 site-specifically to a stabilized dimer of humanized Fab derived from hRS7 (anti-Trop-2), hMN-15 (anti-CEACAM6), and hL243 (anti-HLA-DR), respectively, which were affinity-purified and evaluated in human malignant cell lines of cervix (ME-180), colon (HT-29), esophagus (TE-11, T.Tn), lung (SK-MES-1, A549), liver (HepG2, Huh-7), and skin (A375). Results: As determined by flow cytometry, Trop-2 was highly expressed on the surface of ME-180, TE-11, and T.Tn; moderately on SK-MES-1 and HT-29; and negligibly on HepG2, Huh-7, A549, and A375. In comparison, CEACAM6 was undetectable on SK-MES-1 and A375, but positive on the remaining 7 cell lines, yet at a considerably lower level than Trop-2. Targeting of these immunocytokines to antigen-expressing cells markedly increased the amount of IFN-λ1 localized at the cell surface, as demonstrated by (E1)-λ1 on ME-180 (Trop-2+), (15)-λ1 on HepG2 (CEACAM6+), and (C2)-λ1 on A375 (HLA-DR+), respectively. In MTS assays, (E1)-λ1 inhibited the in vitro proliferation of ME-180, SK-MES-1, and TE-11, with EC50 Citation Format: Donglin Liu, Edmund A. Rossi, Thomas M. Cardillo, David M. Goldenberg, Chien-Hsing Chang. Targeting interferon-λ1 with a stabilized dimer of anti-tumor Fab . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1221. doi:10.1158/1538-7445.AM2013-1221