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Antibody-drug conjugates (ADCs) are cancer chemotherapeutics that utilize a monoclonal antibody (mAb)-based delivery system, a cytotoxic payload, and a chemical linker. ADC payloads must be strategically functionalized to allow linker attachment without perturbing the potency required for ADC efficacy. We previously developed a biocatalytic system for the precise functionalization of tiancimycin (TNM)-based payloads. The TNMs are anthraquinone-fused enediynes (AFEs) and have yet to be translated into the clinic. Herein, we report the translation of biocatalytically functionalized TNMs into ADCs in combination with the dual-variable domain (DVD)-mAb platform. The DVD enables both site-specific conjugation and a plug-and-play modularity for antigen-targeting specificity. We evaluated three linker chemistries in terms of TNM-based ADC potency and antigen selectivity, demonstrating a trade-off between potency and selectivity. This represents the first application of AFE-based payloads to DVDs for ADC development, a workflow that is generalizable to further advance AFE-based ADCs for multiple cancer types.

Site-specifically modified radioimmunoconjugates exhibit superior in vitro and in vivo behavior compared to analogues synthesized via traditional stochastic methods. However, the development of approaches to site-specific bioconjugation that combine high levels of selectivity, simple reaction conditions, and clinical translatability remains a challenge. Herein, we describe a novel solution to this problem: the use of dual-variable domain immunoglobulins (DVD-IgG). More specifically, we report the synthesis, in vitro evaluation, and in vivo validation of a (177)Lu-labeled radioimmunoconjugate based on (HER2)DVD, a DVD-IgG containing the HER2-targeting variable domains of trastuzumab and the catalytic variable domains of IgG h38C2. To this end, we first modified (HER2)DVD with a phenyloxadiazolyl methlysulfone-modified variant of the chelator CHX-A″-DTPA (PODS-CHX-A″-DTPA) and verified the site-specificity of the conjugation for the reactive lysines within the catalytic domains via chemical assay, MALDI-ToF mass spectrometry, and SDS-PAGE. The chelator-bearing immunoconjugate was subsequently labeled with [(177)Lu]Lu(3+) to produce the completed radioimmunoconjugate — [(177)Lu]Lu-CHX-A″-DTPA(PODS)-(HER2)DVD — in >80% radiochemical conversion and a specific activity of 29.5 ± 7.1 GBq/μmol. [(177)Lu]Lu-CHX-A″-DTPA(PODS)-(HER2)DVD did not form aggregates upon prolonged incubation in human serum, displayed 87% stability to demetallation over a 7 d incubation in serum, and exhibited an immunoreactive fraction of 0.95 with HER2-coated beads. Finally, we compared the pharmacokinetic profile of [(177)Lu]Lu-CHX-A″-DTPA(PODS)-(HER2)DVD to that of a (177)Lu-labeled variant of trastuzumab in mice bearing subcutaneous HER2-expressing BT-474 human breast cancer xenografts. The in vivo performance of [(177)Lu]Lu-CHX-A″-DTPA(PODS)-(HER2)DVD matched that of (177)Lu-labeled trastuzumab, with the former producing a tumoral activity concentration of 34.1 ± 12.1 %ID/g at 168 h and tumor-to-blood, tumor-to-liver, and tumor-to-kidney activity concentration ratios of 10.5, 9.6, and 21.8 respectively at the same timepoint. Importantly, the DVD-IgG did not exhibit a substantially longer serum half-life than the traditional IgG despite its significantly larger size (202 kDa for the former vs. 148 kDa for the latter). Taken together, these data suggest that DVD-IgGs represent a viable platform for the future development of highly effective site-specifically labeled radioimmunoconjugates for diagnostic imaging, theranostic imaging, and radioimmunotherapy.

Background In vitro display technologies have been successfully utilized for the discovery and evolution of monoclonal antibodies (mAbs) for diagnostic and therapeutic applications, with phage display and yeast display being the most commonly used platforms due to their simplicity and high efficiency. As their prokaryotic or lower eukaryotic host organisms typically have no or different post-translational modifications, several mammalian cell-based display and screening technologies for isolation and optimization of mAbs have emerged and are being developed. Methods We report here a novel and useful mammalian cell display platform based on the PiggyBac transposon system to display mAbs in single chain Fab (scFab) format on the surface of HEK293F cells. Results Immune rabbit antibody libraries encompassing ~ 7 × 107 independent clones were generated in an all-in-one transposon vector, stably delivered into HEK293F cells, and displayed as scFab with rabbit variable and human constant domains. After one round of magnetic activated cell sorting (MACS) and two rounds of fluorescence activated cell sorting (FACS), mAbs with high affinity in the subnanomolar range and cross-reactivity to the corresponding human and mouse antigens were identified, demonstrating the power of this platform for antibody discovery. Conclusions We developed a highly efficient mammalian cell display platform based on the PiggyBac transposon system for antibody discovery, which could be further utilized for humanization as well as affinity and specificity maturation.

Adoptive transfer, persistence, and migration of autologous control gene-marked T cells.

Serum chemistry and cytokine levels in control animals.

10 million (A) CD8+ TCM-derived CD19-CAR-T-cells, (B) CD19-CAR-T-cells derived from an unselected T-cell population, or (C) CD8+ TCM-derived ROR1-CAR-T-cells were injected into tumor-free NSG mice. After 24 hours, bone marrow, spleens, and peripheral blood were isolated and analyzed for the presence of T-cells (CD45+/CD4+ or CD45+/CD8+) and the expression of activation markers CD25 and CD69 by flow cytometry.

(A) Cytolytic activity of CD8+ TCM-derived T-cells expressing long/CD28, long/4-1BB, and long/CD28_4-1BB CD19-CARs against K562/CD19, K562, and Raji cells. (B) Cytokine production by CAR-T-cells after co-culturing with Raji cells. Supernatants were obtained after 24 hours for analysis. (C) Proliferation of CD19-CAR-T-cells 72 hours after stimulation with Raji cells by CFSE dye dilution. Numbers above each histogram indicate the number of cell divisions the proliferating subset underwent, and the fraction of T-cells in each gate that underwent {greater than or equal to}4/3/2/1 cell divisions is provided in the upper left of each plot.

Genetic engineering of T cells for adoptive transfer by introducing a tumor-targeting chimeric antigen receptor (CAR) is a new approach to cancer immunotherapy. A challenge for the field is to define cell surface molecules that are both preferentially expressed on tumor cells and can be safely targeted with T cells. The orphan tyrosine kinase receptor ROR1 is a candidate target for T-cell therapy with CAR-modified T cells (CAR-T cells) because it is expressed on the surface of many lymphatic and epithelial malignancies and has a putative role in tumor cell survival. The cell surface isoform of ROR1 is expressed in embryogenesis but absent in adult tissues except for B-cell precursors and low levels of transcripts in adipocytes, pancreas, and lung. ROR1 is highly conserved between humans and macaques and has a similar pattern of tissue expression. To determine if low-level ROR1 expression on normal cells would result in toxicity or adversely affect CAR-T cell survival and/or function, we adoptively transferred autologous ROR1 CAR-T cells into nonhuman primates. ROR1 CAR-T cells did not cause overt toxicity to normal organs and accumulated in bone marrow and lymph node sites, where ROR1-positive B cells were present. The findings support the clinical evaluation of ROR1 CAR-T cells for ROR1+ malignancies and demonstrate the utility of nonhuman primates for evaluating the safety of immunotherapy with engineered T cells specific for tumor-associated molecules that are homologous between humans and nonhuman primates. Cancer Immunol Res; 3(2); 206–16. ©2014 AACR.

Gating strategy for ROR1-expressing B cells in the lymph node.

The use of synthetic chimeric antigen receptors (CAR) to redirect T cells to recognize tumor provides a powerful new approach to cancer immunotherapy; however, the attributes of CARs that ensure optimal in vivo tumor recognition remain to be defined. Here, we analyze the influence of length and composition of IgG-derived extracellular spacer domains on the function of CARs. Our studies demonstrate that CD19-CARs with a long spacer from IgG4 hinge-CH2-CH3 are functional in vitro but lack antitumor activity in vivo due to interaction between the Fc domain within the spacer and the Fc receptor–bearing myeloid cells, leading to activation-induced T-cell death. We demonstrate that in vivo persistence and antitumor effects of CAR-T cells with a long spacer can be restored by modifying distinct regions in the CH2 domain that are essential for Fc receptor binding. Our studies demonstrate that modifications that abrogate binding to Fc receptors are crucial for CARs in which a long spacer is obligatory for tumor recognition as shown here for a ROR1-specific CAR. These results demonstrate that the length and composition of the extracellular spacer domain that lacks intrinsic signaling function can be decisive in the design of CARs for optimal in vivo activity. Cancer Immunol Res; 3(2); 125–35. ©2014 AACR.

Supplemental Materials and Methods

(A) Mice were inoculated with 0.5 million Raji-ffluc cells via tail vein injection and tumor engraftment was confirmed by bioluminescence imaging on day six. On day seven, mice received a single i.v. injection of various doses of CD8+ TCM-derived T-cells transduced with the short spacer CD19/4-1BB/CD3ζ-CAR or of control T-cells (EGFRt). (B) Tumor growth was measured by bioluminescence imaging and is plotted for individual mice (4 mice per group). Arrows mark the day of T-cell transfer. Bioluminescence images from day six (one day before T-cell transfer) and day 19 (12 days after transfer) are shown.

PDF File - 122K, Supplementary Figure 5: Expression of ROR1 and NKG2D ligands on epithelial cancer cell lines.

PDF File - 169K, Supplementary Figure 4: The function of ROR1-CAR and CD19-CAR modified CD8+ T-cells against primary CLL is augmented by CAR-modified CD4+ helper T-cells.

PDF File - 125K, Supplementary Figure 3: In vitro function of T-cells expressing CD19-CARs with IgG4-Fc Hinge vs. CD8a Hinge spacer and a signaling module with 4-1BB costimulatory domain.

PDF File - 129K, Supplementary Figure 1: Design of ROR1-CARs with modified spacer length and derived from the 2A2 and R12 scFV with different affinity.

Purpose: The adoptive transfer of T cells modified to express a chimeric antigen receptor (CAR) comprised of an extracellular single-chain antibody (scFV) fragment specific for a tumor cell surface molecule, and linked to an intracellular signaling module, has activity in advanced malignancies. The receptor tyrosine kinase–like orphan receptor 1 (ROR1) is a tumor-associated molecule expressed in prevalent B-lymphoid and epithelial cancers and is absent on normal mature B cells and vital tissues, making it a candidate for CAR T-cell therapy.Experimental Design: We constructed ROR1-CARs from scFVs with different affinities and containing extracellular IgG4-Fc spacer domains of different lengths, and evaluated the ability of T cells expressing each CAR to recognize ROR1+ hematopoietic and epithelial tumors in vitro, and to eliminate human mantle cell lymphoma (MCL) engrafted into immunodeficient mice.Results: ROR1-CARs containing a short “Hinge-only” extracellular spacer conferred superior lysis of ROR1+ tumor cells and induction of T-cell effector functions compared with CARs with long “Hinge-CH2-CH3” spacers. CARs derived from a higher affinity scFV conferred maximum T-cell effector function against primary CLL and ROR1+ epithelial cancer lines in vitro without inducing activation-induced T-cell death. T cells modified with an optimal ROR1-CAR were equivalently effective as CD19-CAR–modified T cells in mediating regression of JeKo-1 MCL in immunodeficient mice.Conclusions: Our results show that customizing spacer design and increasing affinity of ROR1-CARs enhances T-cell effector function and recognition of ROR1+ tumors. T cells modified with an optimized ROR1-CAR have significant antitumor efficacy in a preclinical model in vivo, suggesting they may be useful to treat ROR1+ tumors in clinical applications. Clin Cancer Res; 19(12); 3153–64. ©2013 AACR.

PDF File - 83K, Supplementary Figure 2: Analysis of cytokine production and proliferation of CD4+ T-cells lines modified with a ROR1-CAR derived from mAb R12 with higher affinity than 2A2.

PDF File - 102K, Legend for Supplementary Figures 1-5

Supplementary Figure 4 shows cell surface expression of FcµR on PBMC samples from treatment naïve and relapsed or refractory CLL patients.

Inhibition of tumor angiogenesis is a promising approach for cancer therapy. As an endothelial cell–specific receptor kinase expressed almost exclusively on the surface of vascular endothelium, Tie-2 has an important role in tumor angiogenesis. To explore the therapeutic potential of blocking Tie-2 receptor-interaction pathway, an adenoviral vector was used to deliver a recombinant single-chain antibody fragment rabbit intrabody (pAd-2S03) capable of inhibition of both mouse and human Tie-2 surface expression. pAd-2S03 was given to mice with well-established primary tumors, either a human Kaposi's sarcoma (SLK) or a human colon carcinoma (SW1222). The intrabody significantly inhibited growth of both tumors (75% and 63%, respectively) when compared with pAd-GFP control-treated tumors (P < 0.01). Histopathologic analysis of cryosections taken from mice treated with pAd-2S03 revealed a marked decrease in vessel density, which was reduced by >87% in both tumor models when compared with control-treated tumors (P < 0.01). In contrast, human Tie-2-monospecific pAd-1S05 intrabody did not affect the growth of tumors, indicating that the antitumor effect of pAd-2S03 was due to the inhibition of tumor angiogenesis in these murine models. Our results show that the Tie-2 receptor pathway is essential for both SLK sarcoma and SW1222 colon carcinoma xenograft growth. The present study shows the potential utility of antiangiogenic agents that target the endothelium-specific receptor Tie-2 for down-regulation or genetic deletion.

This table describes relapsed or refractory patients characteristics.

This table describes treatment naive patients characteristics.

Supplementary Figure 3 shows that unconjugated Fcµ-Sec has no anti-tumor activity.

Supplementary Figure 1 shows Western Blot analysis of purified Fcµ-Sec constructs.

This file describes the supplementary materials and methods.

Supplementary Figure 2 shows the circulatory half-life of Fcµ-Sec in NSG mice.

Supplementary Video 2 from Targeting Tumor Angiogenesis with Adenovirus-Delivered Anti-Tie-2 Intrabody

Supplementary Video 3 from Targeting Tumor Angiogenesis with Adenovirus-Delivered Anti-Tie-2 Intrabody

BackgroundDespite numerous therapeutic options, safe and curative therapy is unavailable for most patients with chronic lymphocytic leukemia (CLL). A drawback of current therapies such as the anti-CD20 monoclonal antibody (mAb) rituximab is the elimination of all healthy B cells, resulting in impaired humoral immunity. We previously reported the identification of a patient-derived, CLL-binding mAb, JML-1, and identified sialic acid-binding immunoglobulin-like lectin-6 (Siglec-6) as the target of JML-1. Although little is known about Siglec-6, it appears to be an attractive target for cancer immunotherapy due to its absence on most healthy cells and tissues.MethodsWe used a target-specific approach to mine for additional patient-derived anti-Siglec-6 mAbs. To assess the therapeutic utility of targeting Siglec-6 in the context of CLL, T cell-recruiting bispecific antibodies (T-biAbs) that bind to Siglec-6 and CD3 were engineered into single-chain variable fragment–Fc and dual-affinity retargeting (DART)–Fc constructs. T-biAbs were evaluated for their activity in vitro, ex vivo, and in vivo.ResultsWe discovered the anti-Siglec-6 mAbs RC-1 and RC-2, which bind with higher affinity than JML-1 yet maintain similar specificity. Both JML-1 and RC-1 T-biAbs were effective at activating T cells and killing Siglec-6+target cells. The RC-1 clone in the DART–Fc format was the most potent T-biAb tested and was the only anti-Siglec-6 T-biAb that eliminated Siglec-6+primary CLL cells via autologous T cells at pathological T-to-CLL cell ratios. Tested at healthy T-to-B cell ratios, it also eliminated a Siglec-6+fraction of primary B cells from healthy donors. The subpicomolar potency of the DART–Fc format was attributed to the reduction in the length and flexibility of the cytolytic synapse. Furthermore, the RC-1 T-biAb was effective at clearing MEC1 CLL cells in vivo and demonstrated a circulatory half-life of over 7 days.ConclusionSiglec-6-targeting T-biAbs are highly potent and specific for eliminating Siglec-6+leukemic and healthy B cells while sparing Siglec-6−healthy B cells, suggesting a unique treatment strategy for CLL with diminished suppression of humoral immunity. Our data corroborate reports that T-biAb efficacy is dependent on synapse geometry and reveal that synapse architecture can be tuned via antibody engineering. Our fully human anti-Siglec-6 antibodies and T-biAbs have potential for cancer immunotherapy.Trial registration numberNCT00923507.

Among antibody-based cancer therapies, bispecific antibodies (biAbs) have gained momentum in preclinical and clinical investigations following the regulatory approvals of the trailblazing T-cell engaging biAb (T-biAb) blinatumomab. Discussed herein are recent strategies that aim at boosting the potency and mitigating the toxicity of T-biAbs, broadening their therapeutic utility from hematologic to solid malignancies, and generating T-biAbs in situ. In cancer immunotherapy, T-biAbs are facing fierce competition with chimeric antigen receptor T cells (CAR-Ts), a battle for clinical and commercial viability that will be closely watched. However, innovative combinations of T-biAbs and CAR-Ts have also transpired. NK-cell engaging biAbs (NK-biAbs) are reemerging as an alternative that addresses liabilities of T-biAbs. Beyond NK-biAbs, other biAbs designed to recruit cellular and molecular components of the innate immune system will be covered in this reflection on new tools, technologies, and targets.

The enediynes are among the most cytotoxic molecules known, and their use as anticancer drugs has been successfully demonstrated by targeted delivery. Clinical advancement of the anthraquinone-fused enediynes has been hindered by their low titers and lack of functional groups to enable the preparation of antibody-drug conjugates (ADCs). Here we report biochemical and structural characterization of TnmH from the tiancimycin (TNM) biosynthetic pathway, revealing that (i) TnmH catalyzes regiospecific methylation at the C-7 hydroxyl group, (ii) TnmH exhibits broad substrate promiscuity toward hydroxyanthraquinones and S-alkylated SAM analogues and catalyzes efficient installation of reactive alkyl handles, (iii) the X-ray crystal structure of TnmH provides the molecular basis to account for its broad substrate promiscuity, and (iv) TnmH as a biocatalyst enables the development of novel conjugation strategies to prepare antibody-TNM conjugates. These findings should greatly facilitate the construction and evaluation of antibody-TNM conjugates as next-generation ADCs for targeted chemotherapy.

Objectives of the Study: Bispecific antibodies (BsAbs) are a class of immunotherapy drugs that facilitate targeted killing of cancer cells. Here, we report therapeutic potential of a novel Siglec-6/CD3 targeted bsAb against chronic lymphocytic leukemia (CLL) cells. We show here for the first time the functional and therapeutic relevance of Siglec-6 in CLL cell adhesion and migration. Mechanistically, we have shown Siglec-6 mediated cell adhesion and migration through Siglec-6 ligand (sialyl Tn) mediated DOCK8 dependent activation of Cdc42 associated with actin polymerization. To evaluate the therapeutic potential of Siglec-6, we generated a human (hu) Siglec-6 transgenic mouse model and crossed it with the TCL1 CLL mouse model to obtain Siglec-6 x TCL1 mice which develop Siglec-6+ leukemia. A pilot in-vivo study with anti-Siglec-6/CD3 targeted bsAb showed a survival benefit in humanized CD3 (huCD3) mice engrafted with Siglec-6+ leukemic cells. Methods used: Flow cytometry was used to analyze cell surface expression of Siglec-6 and sialyl Tn (sTn). Mass spectrometry analysis was performed to identify Siglec-6 interacting proteins. CRISPR/Cas9 technique was used to generate knock-out (KO) cell lines for mechanistic studies. Phalloidin staining followed by confocal imaging was used to examine actin polymerization. For in-vivo BsAb treatment experiments, huCD3 mice were engrafted with 5 million Siglec-6+ leukemic cells isolated from Siglec-6 x TCL1 mouse splenocytes. Weekly treatment (i.v) was started after mice display 5% circulating leukemia. Results and Conclusion: Compared to Siglec-6+ CLL cells, Siglec-6- CLL cells exhibited significant reduction in adhesion to (~50%) and migration towards (~50%) CLL-BMSCs. Importantly, a Siglec-6 targeted antibody inhibited homing of Siglec-6+ MEC1 cells and primary CLL cells to the spleen and bone marrow in NSG mice (~35%). Mass spectrometry and co-immunoprecipitation analysis in MEC1 cells revealed interaction of Siglec-6 with DOCK8, a guanine nucleotide exchange factor. Stimulation of Siglec-6+ MEC1 cells with sTn resulted in Cdc42 activation and WASP protein recruitment, followed by increased actin polymerization, that were compromised in Siglec-6 or DOCK8 KO MEC1 cells. Siglec-6+ leukemia engrafted huCD3 mice treated with anti-Siglec-6/CD3 BsAb has so far demonstrated a survival benefit with median survival of 18 weeks versus a median survival of 8 weeks in the control group that received a non-targeting BsAb (n=4/group). Significance: We have for the first time shown Siglec-6 dependent recruitment of DOCK8 leading to migration and adhesion of B-CLL cells. An anti-Siglec-6/CD3 BsAb provides a survival benefit against Siglec-6+ leukemia, paving the way for development of Siglec-6 targeted therapeutics to treat CLL. Citation Format: Jessica Nunes, Charlene Mao, Matthew Purcell, Elizabeth Perry, Liwen Zhang, Xiaokui Mo, Matthew Cyr, Meixiao Long, John Byrd, Christoph Rader, Natarajan Muthusamy. Exploring the therapeutic potential of a novel Siglec-6 targeted bispecific antibody to selectively kill leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2966.

Comparative analyses of four anthraquinone-fused enediyne biosynthetic gene clusters (BGCs) identified YpmL, a cytochrome P450 enzyme, unique to the yangpumicin (YPM) BGC. In vitro characterization of YpmL established it as a hydroxylase, catalyzing C-6 hydroxylation in YPM A biosynthesis. In vivo application of YpmL enabled engineered production of four new tiancimycin analogues (14-17). Evaluation of their cytotoxicity against selected human cancer cell lines shed new insights into enediyne structure-activity relationship.

Background While all clinically translated antibody-drug conjugates (ADCs) contain a single-drug payload, most systemic cancer chemotherapies involve use of a combination of drugs. These regimens improve treatment outcomes and slow development of drug resistance. We here report the generation of an ADC with a dual-drug payload that combines two distinct mechanisms of action. Methods Virtual DNA crosslinking agent PNU-159682 and tubulin polymerization inhibitor monomethyl auristatin F (MMAF) were conjugated to a HER2-targeting antibody via site-specific conjugation at engineered selenocysteine and cysteine residues (thio-selenomab). Results The dual-drug ADC showed selective and potent cytotoxicity against HER2-expressing cell lines and exhibited dual mechanisms of action consistent with the attached drugs. While PNU-159682 caused S-phase cell cycle arrest due to its DNA-damaging activity, MMAF simultaneously inhibited tubulin polymerization and caused G2/M-phase cell cycle arrest. Conclusion The thio-selenomab platform enables the assembly of dual-drug ADCs with two distinct mechanisms of action.

Chronic lymphocytic leukemia (CLL) occurs in 2 major forms: aggressive and indolent. Low miR-29b expression in aggressive CLL is associated with poor prognosis. Indiscriminate miR-29b overexpression in the B-lineage of mice causes aberrance, thus warranting the need for selective introduction of miR-29b into B-CLL cells for therapeutic benefit. The oncofetal antigen receptor tyrosine kinase orphan receptor 1 (ROR1) is expressed on malignant B-CLL cells, but not normal B cells, encouraging us with ROR1-targeted delivery for therapeutic miRs. Here, we describe targeted delivery of miR-29b to ROR1+ CLL cells leading to downregulation of DNMT1 and DNMT3A, modulation of global DNA methylation, decreased SP1, and increased p21 expression in cell lines and primary CLL cells in vitro. Furthermore, using an Eμ-TCL1 mouse model expressing human ROR1, we report the therapeutic benefit of enhanced survival via cellular reprograming by downregulation of DNMT1 and DNMT3A in vivo. Gene expression profiling of engrafted murine leukemia identified reprogramming of cell cycle regulators with decreased SP1 and increased p21 expression after targeted miR-29b treatment. This finding was confirmed by protein modulation, leading to cell cycle arrest and survival benefit in vivo. Importantly, SP1 knockdown results in p21-dependent compensation of the miR-29b effect on cell cycle arrest. These studies form a basis for leukemic cell–targeted delivery of miR-29b as a promising therapeutic approach for CLL and other ROR1+ B-cell malignancies.

This review presents some examples of studies using the European rabbit (Oryctolagus cuniculus) that have led to, and continue to, contribute to advancement of understanding of human diseases as well as therapeutics development. In addition, we tabulate FDA-approved rabbit polyclonal and rabbit monoclonal antibodies (mAbs) that are used for diagnostic applications, as well as an overview of some "humanized" or otherwise altered rabbit mAbs that are in initial phase I, II, or advanced to phase III clinical trials. Information about endogenous retriviruses learned from studies of rabbits and other members of the order Lagomorpha are summarized as this knowledge now applies to new therapeutics being developed for several human diseases including Multiple Sclerosis, Type 1 Diabetes and Cancer.

BACKGROUND: ROR1 is a type 1 transmembrane tyrosine kinase receptor that plays a critical role in embryonic and fetal development. ROR1 has been described as a possible oncogene and is expressed in numerous malignancies including TNBC and non-small cell lung cancer (NSCLC). We are conducting a first-in-human trial targeting ROR1 with CAR-T cells in patients with advanced TNBC and NSCLC. The cellular construct employed targets the Ig/Fz portion of the extracellular domain of ROR1 and contains 4-1BB/CD3ζ intracellular signaling domain. The manufacturing process utilizes autologous peripheral blood lymphocytes, separated into CD4 and CD8 subsets, which are independently cultured with anti-CD3/anti-CD28 beads and IL-2, then transduced with a lentiviral vector encoding the ROR1 CAR. The CAR-T cell product is formulated in a 1:1 ratio of CD4+ and CD8+ CAR-T cells. METHODS: This ongoing phase I trial (NCT02706392) is evaluating the safety of administering ROR1 CAR-T cells in escalating doses (3.3x105, 1x106, 3.3x106 and 1x107 cells/kg) following lymphodepletion with cyclophosphamide-containing regimens using a continual reassessment method (CRM) for dose escalation. TNBC patients with adequate organ function and performance status, measurable disease, and tumors expressing ROR1 (>20% by IHC) are eligible for enrollment. Persistence of CAR-T cells in blood, cytokine levels, measures of immunogenicity and multi-parametric flow cytometry are being evaluated at multiple time points. Imaging assessments by RECIST 1.1 are performed day 28 - 90, then at 6 and 12 months, and every 6 months as clinically indicated to estimate efficacy. RESULTS: To date, 4 TNBC patients (age range 38-67) have been enrolled, treated and are evaluable for response. Patients had received prior therapies for metastatic disease (range 3-11). 3 of 4 had visceral metastases. No dose-limiting toxicities, severe neurotoxicity or severe cytokine release syndrome (sCRS) were observed at dose levels 1 and 2. Two patients experienced grade 1 CRS. 2 of 4 patients had evidence of CAR-T cell expansion between days 14 and 20, with peak CD8+ CAR-T up to 232.1 cells/uL. Analysis of surface phenotype revealed upregulation of inhibitory receptors on CAR-T cells at the peak of expansion, confirmed by RNA seq. Post-treatment tumor biopsy in patient with partial response revealed an influx of CD3+ T cells and macrophages suggesting ROR1 CAR-T cell trafficking. Two patients received 2 CAR-T cell infusions. Two patients had confirmed stable disease at 15 weeks and 19 weeks, respectively. One patient has stable disease after 1st CAR-T cell infusion, then confirmed partial response after 2nd CAR-T cell infusion which has persisted for 14 weeks. Results will be updated. CONCLUSIONS: ROR1+ CAR-T cells can be safely transferred, expand in vivo in patients with TNBC. Current efforts are directed at understanding and overcoming the mechanisms that limit homing, persistence, and/or function at tumor sites. The trial is continuing with dose-escalation. Funding provided by 8RO1 CA114536-11 and Juno Therapeutics. Citation Format: Specht JM, Lee SM, Turtle C, Berger C, Balakrishnan A, Srivastava S, Viollet V, Veatch J, Gooley T, Mullane E, Chaney CN, Rader C, Pierce RH, Gottardo R, Maloney DG, Riddell SR. A phase I study of adoptive immunotherapy for ROR1+ advanced triple negative breast cancer (TNBC) with defined subsets of autologous T cells expressing a ROR1-specific chimeric antigen receptor (ROR1-CAR) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-09-13.

Sialic acid-binding immunoglobulin-like lectins (Siglec) regulate immune function via glycan recognition. Here, we show for the first time the functional relevance of Siglec-6 and its ligand sialyl Tn (sTn) in cell adhesion and migration in chronic lymphocytic leukemia (CLL). Siglec-6+ primary B-CLL cells have significantly enhanced adhesion and migration towards sTn+ CLL− bone marrow stromal cells. A Siglec-6 targeted antibody inhibited homing of Siglec-6+ MEC1 cells and B-CLL cells to the spleen and bone marrow in NSG mice. Mass spectrometry analysis in MEC1 cells revealed interaction of Siglec-6 with DOCK8, a guanine nucleotide exchange factor. Stimulation of Siglec-6+ MEC1 cells with sTn resulted in Cdc42 activation, WASP recruitment and enhanced actin polymerization, which were compromised in Siglec-6 or DOCK8 KO MEC1 cells. Additionally, cell fractionation experiments revealed that Siglec-6+ MEC1 cells and B-CLL cells had higher levels of DOCK8 at the cell membrane when compared to MEC1 Siglec-6 KO cells and normal donor B cells respectively, indicating that Siglec-6 may be responsible for tethering DOCK8 to the cell membrane and is important for promoting actin polymerization at the leading cell edge. To study the therapeutic potential of Siglec-6, we generated a Siglec-6+ CLL mouse model (Siglec-6 x TCL1). Ex vivo experiments with a Siglec-6/CD3 targeted bispecific antibody showed specific killing of Siglec-6+ leukemic B cells. In summary, our studies have identified a novel role for Siglec-6 in promoting DOCK8 dependent CLL cell migration and opens up new therapeutic avenues to eliminate Siglec-6+ malignant B cells. Ongoing studies are focused on evaluating in vivo efficacy of Siglec-6/CD3 targeted bispecific antibody. Natarajan Muthusamy: NIH-R21 Grant and Pelotonia Idea grant Jessica Nunes: Pelotonia Graduate Fellowship

The ammosamides (AMMs) are a family of pyrroloquinoline alkaloids that exhibits a wide variety of bioactivities. A biosynthetic gene cluster (BGC) that is highly homologous in both gene content and genetic organization to the amm BGC was identified by mining the Streptomyces uncialis DCA2648 genome, leading to the discovery of a sub-family of new AMM congeners, named ammosesters (AMEs). The AMEs feature a C-4a methyl ester, differing from the C-4a amide functional group characteristic to AMMs, and exhibit modest cytotoxicity against a broad spectrum of human cancer cell lines, expanding the structure–activity relationship for the pyrroloquinoline family of natural products. Comparative analysis of the ame and amm BGCs supports the use of a scaffold peptide as an emerging paradigm for the biosynthesis of the pyrroloquinoline family of natural products. AME and AMM biosynthesis diverges from a common intermediate by evolving the pathway-specific Ame24 O-methyltransferase and Amm20 amide synthetase, respectively. These findings will surely inspire future efforts to mimic Nature's combinatorial biosynthetic strategies for natural product structural diversity.

Antibody-drug conjugates directed against tumor-specific targets have allowed targeted delivery of highly potent chemotherapy to malignant cells while sparing normal cells. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncofetal protein with limited expression on normal adult tissues and is overexpressed on the surface of malignant cells in mantle cell lymphoma, acute lymphocytic leukemia with t(1;19)(q23;p13) translocation, and chronic lymphocytic leukemia. This differential expression makes ROR1 an attractive target for antibody-drug conjugate therapy, especially in malignancies such as mantle cell lymphoma and acute lymphocytic leukemia, in which systemic chemotherapy remains the gold standard. Several preclinical and phase 1 clinical studies have established the safety and effectiveness of anti-ROR1 monoclonal antibody–based therapies. Herein we describe a humanized, first-in-class anti-ROR1 antibody-drug conjugate, huXBR1-402-G5-PNU, which links a novel anti-ROR1 antibody (huXBR1-402) to a highly potent anthracycline derivative (PNU). We found that huXBR1-402-G5-PNU is cytotoxic to proliferating ROR1+ malignant cells in vitro and suppressed leukemia proliferation and extended survival in multiple models of mice engrafted with human ROR1+ leukemia. Lastly, we show that the B-cell lymphoma 2 (BCL2)-dependent cytotoxicity of huXBR1-402-G5-PNU can be leveraged by combined treatment strategies with the BCL2 inhibitor venetoclax. Together, our data present compelling preclinical evidence for the efficacy of huXBR1-402-G5-PNU in treating ROR1+ hematologic malignancies.

SUMMARYThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates infection of cells expressing angiotensin-converting enzyme 2 (ACE2). ACE2 is also the viral receptor of SARS-CoV (SARS-CoV-1), a related coronavirus that emerged in 2002-2003. Horseshoe bats (genus Rhinolophus) are presumed to be the original reservoir of both viruses, and a SARS-like coronavirus, RaTG13, closely related SARS-CoV-2, has been isolated from one horseshoe-bat species. Here we characterize the ability of S-protein receptor-binding domains (RBDs) of SARS-CoV-1, SARS-CoV-2, and RaTG13 to bind a range of ACE2 orthologs. We observed that the SARS-CoV-2 RBD bound human, pangolin, and horseshoe bat (R. macrotis) ACE2 more efficiently than the SARS-CoV-1 or RaTG13 RBD. Only the RaTG13 RBD bound rodent ACE2 orthologs efficiently. Five mutations drawn from ACE2 orthologs of nine Rhinolophus species enhanced human ACE2 binding to the SARS-CoV-2 RBD and neutralization of SARS-CoV-2 by an immunoadhesin form of human ACE2 (ACE2-Fc). Two of these mutations impaired neutralization of SARS-CoV-1. An ACE2-Fc variant bearing all five mutations neutralized SARS-CoV-2 five-fold more efficiently than human ACE2-Fc. These data narrow the potential SARS-CoV-2 reservoir, suggest that SARS-CoV-1 and -2 originate from distinct bat species, and identify a more potently neutralizing form of ACE2-Fc.

Summary Adoptive therapy using chimeric antigen receptor-modified T cells (CAR-T cells) is effective in hematologic but not epithelial malignancies, which cause the greatest mortality. In breast and lung cancer patients, CAR-T cells targeting the tumor-associated antigen receptor tyrosine kinase-like orphan receptor 1 (ROR1) infiltrate tumors poorly and become dysfunctional. To test strategies for enhancing efficacy, we adapted the KrasLSL-G12D/+;p53f/f autochthonous model of lung adenocarcinoma to express the CAR target ROR1. Murine ROR1 CAR-T cells transferred after lymphodepletion with cyclophosphamide (Cy) transiently control tumor growth but infiltrate tumors poorly and lose function, similar to what is seen in patients. Adding oxaliplatin (Ox) to the lymphodepletion regimen activates tumor macrophages to express T-cell-recruiting chemokines, resulting in improved CAR-T cell infiltration, remodeling of the tumor microenvironment, and increased tumor sensitivity to anti-PD-L1. Combination therapy with Ox/Cy and anti-PD-L1 synergistically improves CAR-T cell-mediated tumor control and survival, providing a strategy to improve CAR-T cell efficacy in the clinic.

The only current curative treatment for chronic lymphocytic leukemia (CLL) is allogenic hematopoietic stem cell transplantation. Chimeric antigen receptor treatment targeting CD19 for CLL achieved some complete responses, suggesting the need for alternative or combinational therapies to achieve a more robust response. In this work, we evaluated CAR-T cells specific for Siglec-6, an antigen expressed in CLL, as a novel CAR-T cell treatment for CLL. We found that detection of SIGLEC6 mRNA and Siglec-6 protein is highly restricted to placenta and immune cells in other tissues and it is not expressed in hematopoietic stem cells. We generated CAR-T cells specific for Siglec-6 based on the sequence of the fully human anti-Siglec-6 antibody (JML1), which was identified in a CLL patient that was cured after allo-hematopoietic stem cell transplantation (alloHSCT), and observed that it specifically targeted CLL cells in vitro and in a xenograft mouse model. Interestingly, a short hinge region increased the activity of CAR-T cells to target cells expressing higher Siglec-6 levels but similarly targeted CLL cells expressing lower Siglec-6 levels in vitro and in vivo. Our results identify a novel CAR-T cell therapy for CLL and establish Siglec-6 as a possible target for immunotherapy.

Gene silencing by RNA interference (RNAi) has emerged as a powerful treatment strategy across a potentially broad range of diseases. Tailoring siRNAs to silence genes vital for cancer cell growth and function could be an effective treatment, but there are several challenges which must be overcome to enable their use as a therapeutic modality, among which efficient and selective delivery to cancer cells remains paramount. Attempts to use antibodies for siRNA delivery have been reported but these strategies use either nonspecific conjugation resulting in mixtures, or site-specific methods that require multiple steps, introduction of mutations, or use of enzymes. Here, we report a method to generate antibody–siRNA (1:2) conjugates (ARCs) that are structurally defined and easy to assemble. This ARC platform is based on engineered dual variable domain (DVD) antibodies containing a natural uniquely reactive lysine residue for site-specific conjugation to β-lactam linker-functionalized siRNA. The conjugation is efficient, does not compromise the affinity of the parental antibody, and utilizes chemically stabilized siRNA. For proof-of-concept, we generated DVD-ARCs targeting various cell surface antigens on multiple myeloma cells for the selective delivery of siRNA targeting β-catenin (CTNNB1). A set of BCMA-targeting DVD-ARCs at concentrations as low as 10 nM revealed significant CTNNB1 mRNA and protein knockdown.

Metastatic medullary thyroid cancer (MTC) is a rare but often aggressive thyroid malignancy with a 5-year survival rate of less than 40% and few effective therapeutic options. Adoptive T cell immunotherapy using chimeric antigen receptor (CAR)-modified T cells (CAR Ts) is showing encouraging results in the treatment of cancer, but development is challenged by the availability of suitable target antigens. We identified glial-derived neurotrophic factor (GDNF) family receptor alpha 4 (GFRα4) as a putative antigen target for CAR-based therapy of MTC. We show that GFRα4 is highly expressed in MTC, in parafollicular cells within the thyroid from which MTC originates, and in normal thymus. We isolated two single-chain variable fragments (scFvs) targeting GFRα4 isoforms a and b by antibody phage display. CARs bearing the CD3ζ and the CD137 costimulatory domains were constructed using these GFRα4-specific scFvs. GFRα4-specific CAR Ts trigger antigen-dependent cytotoxicity and cytokine production in vitro, and they are able to eliminate tumors derived from the MTC TT cell line in an immunodeficient mouse xenograft model of MTC. These data demonstrate the feasibility of targeting GFRα4 by CAR T and support this antigen as a promising target for adoptive T cell immunotherapy and other antibody-based therapies for MTC., Graphical Abstract, Metastatic medullary thyroid carcinoma (MTC) is a rare but aggressive malignancy with no curative options. By sequencing mRNA from a patient’s tumor, Siegel and colleagues identified and characterized GFRα4 as a promising tumor target antigen and demonstrated that CAR T immunotherapy targeting this antigen controlled MTC in tumor xenograft models.