Daudi Xenografts Research Articles

Reducing the Kidney Uptake of High Contrast CXCR4 PET Imaging Agents via Linker Modifications

Purpose: The C-X-C chemokine receptor 4 (CXCR4) is highly expressed in many subtypes of cancers, notably in several kidney-based malignancies. We synthesized, labeled, and assessed a series of radiotracers based on a previous high contrast PET imaging radiopharmaceutical [68Ga]Ga-BL02, with modifications to its linker and metal chelator, in order to improve its tumor-to-kidney contrast ratio. Methods: Based on the design of BL02, a piperidine-based cationic linker (BL06) and several anionic linkers (tri-Aad (BL17); tri-D-Glu (BL20); tri-Asp (BL25); and tri-cysteic acid (BL31)) were substituted for the triglutamate linker. Additionally, the DOTA chelator was swapped for a DOTAGA chelator (BL30). Each radiotracer was labeled with 68Ga and evaluated in CXCR4-expressing Daudi xenograft mice with biodistribution and/or PET imaging studies. Results: Of all the evaluated radiotracers, [68Ga]Ga-BL31 showed the most promising biodistribution profile, with a lower kidney uptake compared to [68Ga]Ga-BL02, while retaining the high imaging contrast capabilities of [68Ga]Ga-BL02. [68Ga]Ga-BL31 also compared favorably to [68Ga]Ga-Pentixafor, with superior imaging contrast in all non-target organs. The other anionic linker-based radiotracers showed either equivocal or worse contrast ratios compared to [68Ga]Ga-BL02; however, [68Ga]Ga-BL25 also showed lower kidney uptake, as compared to that of [68Ga]Ga-BL02. Meanwhile, [68Ga]Ga-BL06 had high non-target organ uptake and relatively lower tumor uptake, while [68Ga]Ga-BL30 showed significantly increased kidney uptake and similar tumor uptake values. Conclusions: [68Ga]Ga-BL31 is an optimized CXCR4-targeting radiopharmaceutical with lower kidney retention that has clinical potential for PET imaging and radioligand therapy.

Abstract 5441: Discovery of a novel RAD51 inhibitor SCR-6992 targeting homologous recombination pathways in cancer

Abstract Targeting DNA damage response (DDR) pathways has been proposed as an approach for amplifying tumor-specific replicative lesions. RAD51 plays a central role in the DDR pathway, and thus represents a promising anti-tumor target. We here report the discovery of a series of next generation RAD51 inhibitors that can prevent RAD51 foci formation and particularly, a pre-clinical candidate molecule SCR-6992 with potent anti-tumor efficacies and desirable pre-clinical development drug properties. SCR-6992 exhibits remarkable anti-proliferative potency in human lymphoma Daudi cells in vitro (IC50 = 12 nM) while has no inhibitory effect in normal human cells. The combination of SCR-6992 with chemotherapeutic agents or DDR-targeting drugs produces synergistical anti-cancer response in lymphoma and solid tumor cell lines. Furthermore, SCR-6992 significantly inhibits tumor growth in Daudi xenograft tumor model with the tumor regression observed when dosed orally at 80 mg/kg/day. SCR-6992 also shows acceptable translational drug properties and pharmacokinetic profiles in both rodents and dogs. SCR-6992 was well tolerated in rats and dogs in 14-day dose range finding studies without noted hematological or gastrointestinal toxicities, when dosed up to 200 mg/kg and 40 mg/kg, respectively. Taken together, SCR-6992 demonstrates as an effective RAD51 inhibitor with encouraging anti-tumor activities and favorable development drug properties. The clinical development plans of SCR-6992 have been formulated for hematologic malignancies and solid tumors. Citation Format: Peng Gu, Liting Xue, Ping Chen, Wenjing Li, Ya Geng, Feng Zhou, Guimei Yang, Xiaohong Hou, Chunyan Zhao, Wenqing Yang, Renhong Tang. Discovery of a novel RAD51 inhibitor SCR-6992 targeting homologous recombination pathways in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5441.

Development of CD19 CAR Engineered Human Placental CD34 +-Derived Natural Killer Cells (CAR19-CYNK) As an Allogeneic Cancer Immunotherapy

Development of CD19 CAR Engineered Human Placental CD34 +-Derived Natural Killer Cells (CAR19-CYNK) As an Allogeneic Cancer Immunotherapy

Small Molecule Activators of Protein Phosphatase 2A (SMAPs) Cause c-Myc Degradation and Tumor Growth Suppression in Preclinical Models of Lymphoid Malignancies

Introduction: c-myc is an oncogene implicated in the initiation and progression of multiple hematologic malignancies. Characteristic translocations of the c-myc gene with the immunoglobulin heavy chain gene (in Burkitt lymphoma and “double-hit” non-Hodgkin lymphoma) and with the T cell receptor gene (in T cell acute lymphoblastic leukemia, T-ALL) lead to constitutive expression of the c-myc transcription factor, ultimately driving dysregulated proliferation (Boxer & Dang, Oncogene 2001; 20(40):5595-5610). Strategies for inhibition of c-myc remain an area of active research; however, to date there are no approved myc-targeted agents within the cancer armamentarium, and cytotoxic chemotherapy remains the standard of care for c-myc-driven lymphoid malignancies. Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase tumor suppressor protein that contributes to c-myc degradation through both direct dephosphorylation of an identified phosphodegron as well as by indirect mechanisms (Farrell & Sears, Cold Spring Harbor Perspect Med 2014; 4(3):1-15). Our laboratory has developed a series of first in class, orally bioavailable small molecule activators of PP2A (SMAPs) by derivatizing tricyclic antipsychotics to maximize antiproliferative activity and minimize psychotropic properties (Sangodkar et al., J Clin Invest 2017;127(6):2081-2090). We hypothesized that activation of PP2A with SMAPs would lead to c-myc degradation and tumor growth inhibition in models of c-myc-driven lymphoid malignancies.Methods: We investigated the effects of SMAP treatment on c-myc content, c-myc half-life (t1/2), cell viability, and xenograft tumor growth in Burkitt lymphoma (BL) and T-ALL cell lines with wild type and mutant c-myc phosphodegrons.Results: SMAP treatment induced apoptosis in Daudi (BL) cells, as evidenced by decreased viability and increased PARP and caspase cleavage. In Daudi (BL) and CCRF-CEM (T-ALL) cell lines, SMAP treatment led to rapid loss of phosphorylated and total c-myc protein. Cycloheximide (CHX)-chase experiments performed in CCRF-CEM cells confirmed that SMAP treatment affects c-myc degradation without affecting protein synthesis. This effect was abrogated by Ser to Glu mutation of residue 62 within the c-myc phosphodegron, suggesting that SMAP-mediated c-myc degradation is PP2A-dependent. Treatment of NOD/SCID mice bearing Daudi xenografts with twice daily oral SMAP decreased tumor c-myc content and inhibited tumor growth without obvious toxicity. Xenografts overexpressing c-myc with phosphodegron mutations (S62D, T58I) were resistant to SMAP treatment.Conclusions: These in vitro and in vivo observations establish PP2A activation as a potential pharmacologic strategy to target c-myc in cancers such as BL, T-ALL, and “double hit” non-Hodgkin lymphoma. SMAPs are well-tolerated, orally bioavailable activators of PP2A poised for further exploration in the treatment of c-myc-driven malignancies, either alone or in combination with other agents. Sequencing of the c-myc phosphodegron prior to initiation of treatment may facilitate patient selection in future clinical trials of PP2A activators. [Display omitted] DisclosuresNarla:Mount Sinai School of Medicine: Patents & Royalties: patents on the small molecule PP2A activators (GN).

High-Contrast CXCR4-Targeted 18F-PET Imaging Using a Potent and Selective Antagonist.

C-X-C chemokine receptor 4 (CXCR4) is highly expressed in cancers, contributing to proliferation, metastasis, and a poor prognosis. The noninvasive imaging of CXCR4 can enable the detection and characterization of aggressive cancers with poor outcomes. Currently, no 18F-labeled CXCR4 positron emission tomography (PET) radiotracer has demonstrated imaging contrast comparable to [68Ga]Ga-Pentixafor, a CXCR4-targeting radioligand. We, therefore, aimed to develop a high-contrast CXCR4-targeting radiotracer by incorporating a hydrophilic linker and trifluoroborate radioprosthesis to LY2510924, a known CXCR4 antagonist. A carboxy-ammoniomethyl-trifluoroborate (PepBF3) moiety was conjugated to the LY2510924-derived peptide possessing a triglutamate linker via amide bond formation to obtain BL08, whereas an alkyne ammoniomethyl-trifluoroborate (AMBF3) moiety was conjugated using the copper-catalyzed [3+2] cycloaddition click reaction to obtain BL09. BL08 and BL09 were radiolabeled with [18F]fluoride ion using 18F-19F isotope exchange. Pentixafor was radiolabeled with [68Ga]GaCl3. Side-by-side PET imaging and biodistribution studies were performed on immunocompromised mice bearing Daudi Burkitt lymphoma xenografts. The biodistribution of [18F]BL08 and [18F]BL09 showed tumor uptake at 2 h postinjection (p.i.) (5.67 ± 1.25%ID/g and 5.83 ± 0.92%ID/g, respectively), which were concordant with the results of PET imaging. [18F]BL08 had low background activity, providing tumor-to-blood, -muscle, and -liver ratios of 72 ± 20, 339 ± 81, and 14 ± 3 (2 h p.i.), respectively. [18F]BL09 behaved similarly, with ratios of 64 ± 20, 239 ± 72, and 17 ± 3 (2 h p.i.), respectively. This resulted in high-contrast visualization of tumors on PET imaging for both radiotracers. [18F]BL08 exhibited lower kidney uptake (2.2 ± 0.5%ID/g) compared to [18F]BL09 (7.6 ± 1.0%ID/g) at 2 h p.i. [18F]BL08 and [18F]BL09 demonstrated higher tumor-to-blood, -muscle, and -liver ratios compared to [68Ga]Ga-Pentixafor (18.9 ± 2.7, 95.4 ± 36.7, and 5.9 ± 0.7 at 2 h p.i., respectively). In conclusion, [18F]BL08 and [18F]BL09 enable high-contrast visualization of CXCR4 expression in Daudi xenografts. Based on high tumor-to-organ ratios, [18F]BL08 may prove a valuable new tool for CXCR4-targeted PET imaging with potential for translation. The use of a PepBF3 moiety is a new approach for the orthogonal conjugation of organotrifluoroborates for 18F-labeling of peptides.

A new class of PentixaFor- and PentixaTher-based theranostic agents with enhanced CXCR4-targeting efficiency.

Non-invasive PET imaging of CXCR4 expression in cancer and inflammation as well as CXCR4-targeted radioligand therapy (RLT) have recently found their way into clinical research by the development of the theranostic agents [68Ga]PentixaFor (cyclo(D-Tyr1-D-[NMe]Orn2(AMBS-[68Ga]DOTA)-Arg3-Nal4-Gly5) = [68Ga]DOTA-AMBS-CPCR4) and [177Lu/90Y]PentixaTher (cyclo(D-3-iodo-Tyr1-D-[NMe]Orn2(AMBS-[177Lu/90Y]DOTA)-Arg3-Nal4-Gly5) = [177Lu/90Y]DOTA-AMBS-iodoCPCR4). Although convincing clinical results have already been obtained with both agents, this study was designed to further investigate the required structural elements for improved ligand-receptor interaction for both peptide cores (CPCR4 and iodoCPCR4). To this aim, a series of DOTA-conjugated CPCR4- and iodoCPCR4-based ligands with new linker structures, replacing the AMBA-linker in PentixaFor and PentixaTher, were synthesized and evaluated.Methods: The in vitro investigation of the novel compounds alongside with the reference peptides PentixaFor and PentixaTher encompassed the determination of hCXCR4 and mCXCR4 affinity (IC50) of the respective natGa-, natLu-, natY- and natBi-complexes in Jurkat and Eμ-myc 1080 cells using [125I]FC-131 and [125I]CPCR4.3 as radioligands, respectively, as well as the evaluation of the internalization and externalization kinetics of selected 68Ga- and 177Lu-labeled compounds in hCXCR4-transfected Chem-1 cells. Comparative small animal PET imaging studies (1h p.i.) as well as in vivo biodistribution studies (1, 6 and 48h p.i.) were performed in Daudi (human B cell lymphoma) xenograft bearing CB17 SCID mice.Results: Based on the affinity data and cellular uptake studies, [68Ga/177Lu]DOTA-r-a-ABA-CPCR4 and [68Ga/177Lu]DOTA-r-a-ABA-iodoCPCR4 (with r-a-ABA = D-Arg-D-Ala-4-aminobenzoyl-) were selected for further evaluation. Both analogs show app. 10-fold enhanced hCXCR4 affinity compared to the respective references [68Ga]PentixaFor and [177Lu]PentixaTher, four times higher cellular uptake in hCXCR4 expressing cells and improved cellular retention. Unfortunately, the improved in vitro binding and uptake characteristics of [68Ga]DOTA-r-a-ABA-CPCR4 and -iodoCPCR4 could not be recapitulated in initial PET imaging studies; both compounds showed similar uptake in the Daudi xenografts as [68Ga]PentixaFor, alongside with higher background accumulation, especially in the kidneys. However, the subsequent biodistribution studies performed for the corresponding 177Lu-labeled analogs revealed a clear superiority of [177Lu]DOTA-r-a-ABA-CPCR4 and [177Lu]DOTA-r-a-ABA-iodoCPCR4 over [177Lu]PentixaTher with respect to tumor uptake (18.3±3.7 and 17.2±2.0 %iD/g, respectively, at 1h p.i. vs 12.4±3.7%iD/g for [177Lu]PentixaTher) as well as activity retention in tumor up to 48h. Especially for [177Lu]DOTA-r-a-ABA-CPCR4 with its low background accumulation, tumor/organ ratios at 48h were 2- to 4-fold higher than those obtained for [177Lu]PentixaTher (except for kidney).Conclusions: The in-depth evaluation of a series of novel CPCR4- and iodoCPCR4 analogs with modified linker structure has yielded reliable structure-activity relationships. It was generally observed that a) AMBA-by-ABA-substitution leads to enhanced ligand internalization, b) the extension of the ABA-linker by two additional amino acids (DOTA-Xaa2-Xaa1-ABA-) provides sufficient linker length to minimize the interaction of the [M3+]DOTA-chelate with the receptor, and that c) introduction of a cationic side chain (Xaa2) greatly enhances receptor affinity of the constructs, obliterating the necessity for Tyr1-iodination of the pentapeptide core to maintain high receptor affinity (such as in [177Lu]PentixaTher).As a result, [177Lu]DOTA-r-a-ABA-CPCR4 has emerged from this study as a powerful second-generation therapeutic CXCR4 ligand with greatly improved targeting efficiency and tumor retention and will be further evaluated in preclinical and clinical CXCR4-targeted dosimetry and RLT studies.

Development of CD38 CAR Engineered Human Placental Hematopoietic Stem Cell Derived Natural Killer Cells (PNK-CAR38) As Allogeneic Cancer Immunotherapy

Background: Natural killer (NK) cells exhibit innate anti-tumor activity owing to the expression of a multitude of activating receptors. Celularity has developed a GMP process for generating off-the-shelf and allogeneic human Placental Hematopoietic Stem Cell derived Natural Killer (PNK-007) cells. To develop a tumor targeted approach, we have engineered PNK cells to express Chimeric Antigen Receptor (CAR) against tumor antigen CD38. PNK cells expressing CD38 CAR (PNK-CAR38) have enhanced anti-tumor function against CD38+ lymphoma and multiple myeloma (MM) cell lines in pre-clinical studies and do not exhibit on-target off-tumor cytotoxicity against healthy CD38+ cells. Here we report the development of a CD38 CAR specific NK cell therapy. Methods: PNK-CAR38 cells were generated through transduction of human placental CD34+ cells with a retroviral vector carrying second generation anti-CD38 chimeric antigen receptor (CAR2-anti-CD38 A2; CD38scFv-CD28CD3ζ) followed by expansion and differentiation of transduced CD34+ cells into NK cells in presence of cytokines including thrombopoietin, SCF, Flt3 ligand, IL-7, IL-15 and IL-2. The anti-tumor activity of untransduced PNK and PNK-CAR38 cells was assessed against Lymphoma (Daudi and Raji) and Multiple Myeloma (Molp8, LP1 and OPM2) cell lines at various effector to target (E:T) ratios. PNK-CAR38 in vivo anti-tumor activity was assessed in a disseminated lymphoma xenograft model in NSG mice. Briefly, luciferase-expressing Daudi cells (3×106) were intravenously (IV) injected at Day0, followed by IV injection of PNK-CAR38 cells (20×106) at Day1 and Day3. Tumor burden was assessed weekly by Bioluminescence Imaging (BLI). Differences between the groups were calculated using two-tailed Student t-test. Results: Placental CD34+ cells were efficiently gene-modified using a retroviral vector and achieved a median of 64% (range 38% to 87%; n=11 donors) CD38 CAR expression in PNK cells at the end of 35-day culture. The median PNK-CAR38 cell yield achieved was 29,000-fold (range 7,800-48,000 fold; n=8) with NK purity >90% measured by CD3-CD56+ population. In vitro an overall improvement in anti-tumor response by PNK-CAR38 over PNK cells was observed. In a NK resistant lymphoma cell line model Daudi, PNK-CAR38 cells from various donors (n=8) elicited 72%±10% lysis versus 6%±3.5% lysis by PNK alone (p<0.01) at 10:1 E:T ratio. PNK-CAR38 elicited significant cytotoxicity 27%±9% versus 13%±5% by PNK cells (p<0.01) against Raji cells depending on NK cell donor. Using MM lines, at 10:1 E:T ratio, PNK-CAR38 cells elicited 33%±16% lysis versus 21%±12% by PNK (p<0.01) against LP1; 44%±6% versus 28%±3% by PNK (p<0.01) against OPM2; and 32%±6% versus 2%±0.5% by PNK (p<0.01) against MOLP8. Since CD38 is also expressed on normal immune and progenitor cells, PNK-CAR38 cells were co-cultured with healthy donor-derived activated T cells and CD34+CD38+ progenitor cells. No on-target off-tumor cytotoxicity against these cells was observed. In the disseminated Daudi xenograft NSG model, PNK-CAR38 significantly reduced tumor burden (approximately 50% BLI reduction) compared to vehicle control 10 days post treatment. Conclusions: The novel PNK-CAR38 demonstrated enhanced in vitro cytotoxic potential against lymphoma and MM lines and showed no on-target off-tumor cytotoxicity against healthy donor-derived cells in vitro. In vivo anti-tumor activity of PNK-CAR38 was shown in a lymphoma disseminated NSG model. Further development of PNK-CAR38 for hematological malignancies is warranted. Disclosures Somanchi: Celularity Inc: Employment. Guo:Celularity, Inc.: Employment. He:Celularity Inc: Employment. Mathur:Celularity Inc: Employment. Difiglia:Celularity Inc: Employment. Rana:Celularity Inc: Employment. Ling:Celularity Inc: Employment. Edinger:Celularity Inc: Employment. Kaufmann:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Zeldis:Sorrento Therapeutics Inc: Employment, Equity Ownership. Ji:Sorrento Therapeutics Inc: Employment, Equity Ownership, Patents & Royalties; Celularity, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Hariri:Celularity Inc: Employment. Zhang:Celularity Inc: Employment.

Engineering High Affinity and Cleavage Resistant CD16 to Augment ADCC of Placental Hematopoietic Stem Cells-Derived Natural Killer Cells

Background: Celularity, Inc. is developing human placental hematopoietic stem cells-derived, cryopreserved, off-the-shelf, ex-vivo expanded and allogenic natural killer (PNK) cells for various hematological malignancies and solid tumors. NK cells play a central role in antibody dependent cell mediated cytotoxicity (ADCC) through Fc receptor CD16 in monoclonal antibody mediated anti-tumor therapies. Two allelic forms of CD16 have been identified. The 158Val/Val form has shown to have higher IgG binding affinity compared to the 158Phe/Phe form.1 The high IgG binding allele are found in about 10-20% of the normal population.2,3 In addition, activation of NK cells induces CD16 shedding by matrix metalloprotease ADAM17 at 197Ser, thus limiting ADCC responses. A single mutation (Ser197Pro) prevents CD16 shedding and increases ADCC activity in NK cells.4 Since the antibody binding affinity and CD16 expression of PNK could vary with different donors, we hypothesize that expressing a high affinity (158Val) and proteinase cleavage resistant (197Pro) CD16 variant (CD16VP) augments anti-tumor ADCC activity. Methods: Lentivirus expressing CD16VP was used to transduce human placental CD34+ cells. After transduction, the cells were cultured in the presence of cytokines including thrombopoietin, SCF, Flt3 ligand, IL-7, IL-15 and IL-2, for 35 days to generate PNK-CD16VP cells. Non-transduced PNK cells (NT) served as a control. Expression of CD16VP was evaluated by activating cells with PMA/ionomycin to induce CD16 cleavage (CD16 shedding assay) followed by immunostaining with CD16 antibody and analyzed using flowcytometry. ADCC of PNK-CD16VP cells was assessed against Daratumumab (anti-CD38) or Rituximab (anti-CD20) opsonized lymphoma cell lines at various effector to target (E:T) ratios. IgG was used as ADCC control. In vivo anti-tumor activity was assessed in a Daudi disseminated Xenograft model in NSG mice. Luciferase-expressing Daudi cells (3x106) were intravenously (IV) administered at day 0, followed by PNK-CD16VP cells (10x106) IV at day 1 and day 3, and Daratumumab at day 3. Tumor burden in mice was monitored by Bioluminescence Imaging (BLI). Statistical differences between the groups were calculated using paired t-test using Prism. Results: Lentiviral transduction of CD16VP achieved high expression efficiency in multiple placental CD34+ donors. These cells expanded [7095 ± 2998 folds (n=8)] and differentiated into PNK cells (>90% CD56+CD3-) at day 35. PNK-CD16VP expressed 64.6 ± 10.3% (n=8) of CD16, while the NT expressed 12.1 ± 3.3% (n=8) CD16. PMA/ionomycin induced >89% shedding of CD16 in NT cells, while significantly less (<11%) CD16 shedding was observed in PNK-CD16VP cells. These results indicated that CD16VP was expressed and maintained throughout the culture process. In vitro ADCC assay demonstrated improved anti-tumor activity of PNK-CD16VP cells over NT cells against Daratumumab or Rituximab opsonized lymphoma cell lines. At 10:1 E:T ratio PNK-CD16VP cells elicited higher cytotoxicity compared to NT: 47 ± 13% against Daratumumab opsonized Daudi cells versus 25 ± 5% (n=5; p<0.05); 30 ± 13% against Daratumumab opsonized HS-Sultan cells versus 21 ± 14% (n=3; p<0.05); 30 ± 7% against Daratumumab opsonized Sudhl6 cells versus 16 ± 10% (n=3; p<0.05). Improved ADCC activities in PNK-CD16VP were also observed in other cell lines including Raji and Sudhl4 with Daratumumab and Rituximab antibodies. PNK-CD16VP were used to test anti-tumor ADCC in vivo using a disseminated Daudi Xenograft model. The preliminary data demonstrated that PNK-CD16VP combined with Daratumumab reduced BLI signal (>50%) compared to vehicle or Daratumumab alone at day 10 after treatment. This observation suggested that PNK-CD16VP demonstrated in vivo ADCC anti-tumor activity. Conclusions: In this study, we genetically modified PNK to express high affinity and cleavage resistant CD16 variant using lentivirus. The PNK-CD16VP cells demonstrated enhanced ADCC function against lymphoma cell lines in vitro and in vivo. Further development of PNK-CD16VP for immune-oncology therapeutics is warranted.

A Novel Class of Bifunctional Immunotherapeutic That Exploits a Universal Antibody Binding Terminus (uABT) to Recruit Endogenous Antibodies to Cell Expressing CD38 Demonstrate In Vivo efficacy in Three Distinct Animal Models

Background: Antibody recruiting molecules (ARM) represent a new modality in immunotherapy of cancer. These are bifunctional molecules composed of two active termini connected by a linker. One of the termini binds to a target molecule on a cancer cell. The other terminus can recruit endogenous IgG antibodies independent of their antigen binding specificity representing a breakthrough improvement to previous approaches (Murelli et al.J Am Chem Soc. 2009). We named this active moiety a universal antibody binding terminus (uABT). As a result of antibody recruitment to the cell surface, the target cell is "opsonized" by antibodies which then bring in the immune effector cells to eliminate the target through various antibody-dependent destruction mechanisms. Multiple myeloma is a neoplasm that arises from terminally differentiated immunoglobulin producing long-lived plasma cells with 32,000 new cases diagnosed each year. Kleo Pharmaceuticals has developed a series of compounds, CD38-ARM which target human CD38 highly expressed by multiple myeloma cells. CD38 -ARM compounds are therapeutically active in three distinct in vivo models without depleting CD38 expressing immune effector cells like existing therapeutic antibodies such as Daratumumab. Methods: CD38-ARM compounds were tested in three independent in vivo models. In the first model, intraperitoneal Daudi xenograft in SCID mice, 20x106 fluorescently labelled Burkitt lymphoma CD38-expressing Daudi cells were injected into SCID mice and treated with 3 mg/kg of compounds. Peritoneal exudates were examined 24 hours later for percentages and absolute numbers of Daudi cells recovered. Activity of compounds was further tested in a MOLP-8 multiple myeloma cell xenograft model in nude mice. Animals received 106 MOLP8 cells subcutaneously, and were treated with a daily dose of 10 mg/kg after tumor volumes of 150 mm3 were reached. Finally, CD38-ARM efficacy was examined in hu IL-15 transgenic NOG mice that have been preconditioned with busulfan and reconstituted i.p. with 2x106human NK cells. Three weeks after reconstitution, 5x106RAJI cells expressing CD38 were implanted s.c. and treatment commenced a week later with 10 mg/kg QDx14. Mice were monitored for NK cell levels and activation status in the blood during the whole study duration by flow cytometry. Results: CD38-uAbt compounds are able to induce clearance of Burkitt's lymphoma Daudi cells expressing high levels of CD38 in a SCID mouse intraperitoneal model. In addition, we show efficacy of one of these compounds in a multiple myeloma xenograft model in nude mice. Using the MOLP8 subcutaneously implanted tumor model we show that administration of our lead molecule is able to induce 50% tumor growth inhibition (TGI) after a daily dosing schedule against this tumor. This activity is comparable to a pharmacologically relevant dose of Daratumumab. Finally, we demonstrate that CD38-ARM treatment shows significant efficacy in humanized mouse model, where IL-15 Tg NOG mice have been reconstituted with human NK cells prior to tumor implantation. In this model daily dosing with the CD38-ARM resulted in up to 70% TGI when compared to untreated control groups. Using this dose/schedule, the molecule did not elicit NK cell depletion as noted in the Daratumumab group. Conclusions: We demonstrated that CD38-ARM compounds are therapeutically active in three distinct in vivo models. Depletion of Daudi cells in a peritoneal SCID model provides strong evidence for the CD38-ARM's capacity to engage macrophage effector functions. Profound inhibition of tumor growth in the NK cell centered humanized hIL-15 Tg NOG mouse model indicates that killing of target cells is executed by human NK cells and provides a foundation to advancing our compounds towards the clinic. Overall, our results demonstrate value of CD38-ARM molecules both as stand-alone therapeutic as well as a platform to develop compounds tailored to a specific indication, by varying target binding moiety of the molecule. Our results also indicate that CD38-ARM compounds engage a variety of effector mechanisms involved in tumor clearance and tumor growth delay, indicating therapeutic potential across a wide range of clinical settings. Disclosures Bunin: Kleo pharmaceuticals: Employment, Equity Ownership. McGrath:Kleo pharmaceuticals: Employment, Equity Ownership. Rossi:Kleo pharmaceuticals: Employment, Equity Ownership. Welsch:Kleo pharmaceuticals: Employment, Equity Ownership. Vidal:Kleo pharmaceuticals: Employment, Equity Ownership. Trinh:Kleo pharmaceuticals: Employment, Equity Ownership. Spiegel:Kleo pharmaceuticals: Equity Ownership. Rastelli:Kleo pharmaceuticals: Employment, Equity Ownership. Alvarez:Kleo pharmaceuticals: Employment, Equity Ownership.

68Ga]Ga/[177Lu]Lu-BL01, a Novel Theranostic Pair for Targeting C-X-C Chemokine Receptor 4.

C-X-C chemokine receptor type 4 (CXCR4) is overexpressed in hematological and solid malignancies. LY2510924 is a potent peptide antagonist of CXCR4. A derivative of LY2510924, BL01, was evaluated for theranostic applications targeting CXCR4. Methods: BL01 was synthesized by solid phase approach. A Lys(ivDde) residue was added at the C-terminus of LY2510924 (cyclo[Phe-Tyr-Lys(iPr)-d-Arg-2-Nal-Gly-d-Glu]-Lys(iPr)-NH2). A DOTA chelator was conjugated to the side chain of the deprotected exogenous Lys residue. The binding affinity of Ga/Lu-BL01 was determined by competitive radioligand binding assays. BL01 was radiolabeled with 68GaCl3 or 177LuCl3. Biodistribution studies were performed in mice bearing Daudi Burkitt's lymphoma tumor xenografts at selected time points. PET imaging studies were performed with [68Ga]Ga-BL01, with blocking experiments performed with preinjection of LY2510924. The stability of [68Ga]Ga/[177Lu]Lu-BL01 was assessed in mouse plasma. Results: Ga-BL01 and Lu-BL01 have nanomolar affinity for CXCR4. [68Ga]Ga-BL01 was obtained in 58 ± 5% decay-corrected radiochemical yields and >99% radiochemical purity with a molar activity of 40 ± 11 GBq/μmol, while [177Lu]Lu-BL01 was obtained in 65 ± 6% decay-corrected radiochemical yields and >99% radiochemical purity with a molar activity of 120 ± 21 GBq/μmol. [68Ga]Ga-BL01 and [177Lu]Lu-BL01 were excreted primarily through the renal pathway. Daudi xenografts were clearly delineated in PET images with good contrast. On the basis of biodistribution data, tumor uptake of [68Ga]Ga-BL01 was 10.2 ± 2.56% injected dose per gram (%ID/g) at 1 h postinjection (p.i.). Spleen (12.6 ± 2.36 %ID/g) and lungs (13.2 ± 2.98 %ID/g), organs that express CXCR4, had high uptake as well. Preinjection of LY2510924 reduced average uptake of [68Ga]Ga-BL01 in tumors by 88%, demonstrating target specificity. The uptake of [68Ga]Ga-BL01 in tumor increased to 15.3 ± 1.86 %ID/g at 2 h p.i., with improved contrast. [177Lu]Lu-BL01 has similar pharmacokinetics as [68Ga]Ga-BL01 at 1 h p.i. The highest uptake was observed in tumor (14.0 ± 1.11 %ID/g), followed by the lungs (13.0 ± 1.27 %ID/g) and spleen (11.6 ± 1.78 %ID/g). The tumor uptake increased to 16.2 ± 2.69 %ID/g at 4 h p.i., before declining slightly to 10.1 ± 1.41 %ID/g at 24 h p.i. Both compounds were stable in vivo, as no metabolites were observed at 5 min p.i. Conclusions: [68Ga]Ga-BL01 and [177Lu]Lu-BL01 are a promising theranostic pair for imaging and endoradiotherapy of CXCR4-expressing malignancies.

Combination of 177 Lu-lilotomab with rituximab significantly improves the therapeutic outcome in preclinical models of non-Hodgkin's lymphoma.

To investigate the therapeutic potential of the next-generation anti-CD37 radioimmunoconjugate 177 Lu-lilotomab satetraxetan (177 Lu-lilotomab) in combination with the anti-CD20 antibody rituximab for treatment of mice with non-Hodgkin's lymphoma (NHL) xenografts. Nude mice with subcutaneous (s.c.) Burkitt's lymphoma Daudi xenografts and SCID mice intravenously (i.v.) injected with Mantle cell lymphoma Rec-1 cells were treated with either 177 Lu-lilotomab or rituximab alone or with the combination of both treatments. Tumour volume, body weight, blood counts and clinical status were monitored. CD20 expression was measured using flow cytometry with fluorescence-labelled rituximab. The combination of 177 Lu-lilotomab and rituximab was synergistic for treatment of nude mice with s.c. Daudi xenografts while it was additive for treatment of SCID mice with i.v. injected Rec-1 cells. Binding of rituximab to NHL cells in-vitro was increased by pretreatment with 177 Lu-lilotomab. Treatment of mice with NHL xenografts with 177 Lu-lilotomab synergistically increased tumour suppression of subsequent anti-CD20 immunotherapy and improved survival. If the same effect is confirmed in a recently started clinical study, it could change the way radioimmunotherapy and CD20 immunotherapy would be used in the future.

First 18F-Labeled Pentixafor-Based Imaging Agent for PET Imaging of CXCR4 Expression In Vivo.

In vivo quantification of CXCR4 expression using [68Ga]pentixafor for positron emission tomography (PET) imaging has gained significant clinical interest as CXCR4 plays a fundamental role in oncology and possesses potential prognostic value when overexpressed. To combine the excellent CXCR4-targeting properties of pentixafor-based tracers with the favorable radionuclide properties of 18F for high-resolution PET imaging, we developed an Al18F-labeled 1,4,7-triazacyclononane-triacetic acid (NOTA) analog of pentixather. Al18F-labeling of NOTA-pentixather was performed in aqueous dimethyl sulfoxide (DMSO) at pH = 4 (105°C, 15 minutes). CXCR4 affinities were determined in competitive binding assays, and both biodistribution and small-animal PET studies were performed in Daudi lymphoma-bearing mice. Under non-optimized conditions, [18F]AlF-NOTA-pentixather was obtained in radiochemical yields of 45.5% ± 13.3% and specific activities of up to 24.8 GBq/μmol. Compared with [natGa]pentixafor, [natF]AlF-NOTA-pentixather showed 1.4-fold higher CXCR4 affinity. [18F]AlF-NOTA-pentixather displayed high and CXCR4-specific in vivo uptake in Daudi xenografts (13.9% ± 0.8% injected dose per gram [ID/g] at 1 hour post injection [p.i.]). Because of its enhanced lipophilicity (logP = −1.4), [18F]AlF-NOTA-pentixather showed increased accumulation in the gall bladder and intestines. However, tumor/background ratios of 7.0 ± 1.2, 2.0 ± 0.3, 2.2 ± 0.4, 16.5 ± 6.5, and 29.2 ± 4 for blood, liver, small intestine, gut, and muscle, respectively, allowed for high-contrast visualization of Daudi tumors using PET (1 hour p.i.). The relatively straightforward radiosynthesis and efficient CXCR4 targeting of [18F]AlF-NOTA-pentixather demonstrate the successful implementation of 18F-complexation chemistry and pentixather-based CXCR4 targeting. Upon pharmacokinetic optimization, this class of tracers holds great promise for future application in humans.

Abstract A15: In vivo efficacy of a CD38-specific engineered toxin body.

Abstract The cell surface glycoprotein CD38 is highly expressed on the surface of several B cell lineage cancers, such as the plasma cell malignancy multiple myeloma, some lymphomas and chronic lymphocytic leukemia (CLL), where it is a marker of unfavorable prognosis. CD38 is a transmembrane receptor and ectoenzyme that is absent or expressed at a low level on most resting leukocytes under normal conditions, making this an appealing target for directed therapy for hematological malignancies. Recent clinical trials with antibodies to CD38 have shown promise in multiple myeloma. While these antibodies rely on the recruitment of an immune response for cytotoxicity, we have developed an engineered toxin body (ETB) comprising a CD38 binding scFv and a modified Shiga-like Toxin A subunit capable of specifically recognizing and directly killing CD38 expressing cells. The CD38 targeted ETB has a different mechanism of action than current treatments for multiple myeloma (such as immunomodulatory agents, protease inhibitors and chemotherapies). Our CD38 targeted ETB has shown to be well tolerated in mice and displays dose dependent efficacy in a CD38 positive tumor setting. The CD38 targeted ETB significantly reduced tumor burden and increased survival over a 40-fold dose range in a disseminated Daudi xenograft, early treatment model. In this setting, the mean tumor burden at day 28 was reduced to 29% of control for the lowest dose group (0.05 mg/kg) and less than 1% of control for the two higher doses (0.5 and 2 mg/kg). Median survival of the control, untreated group was 34 days; the low dose group had extended median survival to 59.5 days and, at the day 60 study end, 90 or 100% of mice were alive in the higher two dosing groups. Our results show that the CD38 targeted ETB is a promising targeted therapeutic agent against CD38 positive cancer cells and is currently under further development. Citation Format: Garrett L. Robinson, Sangeetha Rajagopalan, Brigitte Brieschke, Jennifer Erdman, Jane Neill, Rodney Flores, Julia Foree, William Null, Jensing Liu, Jack P. Higgins, Erin K. Willert. In vivo efficacy of a CD38-specific engineered toxin body. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A15.

Abstract LB-163: Development of an immunoPET tracer for imaging human CD8+ T cells

Abstract Background: Infiltration of CD8+ T cells into human tumors is associated with an increased disease-specific survival in many cancers. A better understanding of T cell trafficking and characterization of the subset of CD8+ T cells with the highest localized activity would improve the ability to schedule and select patients for chemo- and immuno-therapy regimens. To facilitate this objective, we have engineered a humanized anti-CD8 antibody fragment and used it to image human CD8+ T cells in a tumor xenograft and humanized mouse model. Methods: The VH and VL sequences of a murine anti-CD8 antibody were cloned by RT-PCR, engineered into minibody (Mb) fragments (scFv-CH3 dimer) of 80 kDa in size and humanized by CDR grafting onto a human germline framework. Multiple humanized variants were evaluated and the lead Mb candidate was selected based on ELISA, flow cytometry and SPR binding properties. The lead Mb, IAb22G3M1 was transiently expressed in CHO cells and purified by Protein L chromatography. PET imaging was performed with desferrioxamine (Df) conjugated IAb22G3M1 radiolabeled with Zr-89 (T1/2 3.3 d). SCID mice bearing subcutaneous HPB-ALL (CD8+ve) or Daudi (CD8-ve) xenografts were serially imaged at 4, 24 and 41 h after i.v. administration of 89Zr-IAb22G3M2 and tissues harvested and counted to determine the biodistribution at the time of sacrifice. The Mb was also evaluated in NOD-SCID-Gamma (NSG) mice that were engrafted with 20 million human PBMCs. In this latter study IAb22G3M1 was radiolabeled with Cu-64 (T1/2 12.7 hrs) following NODAGA conjugation. Mice were imaged at 4 and 7 hrs followed by tissue collection for biodistribution. NSG mice that were not grafted with PBMCs served as experimental controls. Results: Purification of IAb22G3M1 yielded a product that migrated at the expected MW of 80 kDa with very low (<1%) HMW aggregate. Cell based binding to human CD8 expressing T cells demonstrated a relative affinity of <0.1 nM. Following conjugation and radiolabeling, immunoreactivity was preserved. PET imaging of HPB-ALL xenografts showed specific tumor targeting at 24 and 41 h. The uptake in the CD8 positive HPB-ALL tumors was ∼6-fold higher (10.0±2.4%ID/g) than in the negative Daudi tumors (1.6±0.4%ID/g). Positive tumor to blood ratio was 13.5 for the positive vs. 3.5 for the negative tumor. In mice engrafted with human PBMCs, spleen uptake was 35.8±9.2%ID/g compared to 16.8±3.2%ID/g in control mice. The apparent splenic antigen sink in PBMC grafted mice likely explains the rapid blood clearance and the different tissue uptakes observed between the two groups. Conclusion: We have successfully generated a functional anti-CD8 imaging agent that can be used to detect and monitor T cell trafficking and expansion in vivo. Pre-clinical PET imaging studies suggest that IAb22G3M1 is a promising tracer for detecting CD8 positive immune cells in vivo in suitable models paving the way for clinical translation. Citation Format: Tove Olafsen, David Ho, Eric Epin, Green Zhang, Michael Torgov, Charlie Beigarten, Giti Agahi, Edward Cabral, Anna M. Wu, Jean M. Gudas. Development of an immunoPET tracer for imaging human CD8+ T cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-163. doi:10.1158/1538-7445.AM2014-LB-163

Abstract 4644: Efficacy, pharmacokinetics and tissue distribution of celastrol analogues SBI 064601, SBI 0640599 and SBI 0069272 in C.B-17 SCID mice bearing Daudi Burkitt's lymphoma xenografts

Abstract Introduction: c-Myc plays an important role in cellular proliferation, differentiation, apoptosis and cell cycle progression and its overexpression is associated with aggressiveness and poor prognosis in many cancers. Three celastrol analogues, SBI 064601(601), SBI 0640599(599) and SBI 0069272(272) are potent c-Myc inhibitors. We have evaluated the ability of these celastrol analogues to inhibit Daudi lymphoma cells in vitro and in vivo and characterized the pharmacokinetics of these celastrol analogues in C.B-17 SCID mice bearing Daudi xenografts. Methods: Inhibition of Daudi cell growth by three celastrol analogues was assessed by MTT assay after 72 h of incubation. For the efficacy study, the mice bearing Daudi xenografts were treated with 20 mg/kg iv of 601, 599, 272 qdx5, or with 50 mg/kg of 601(iv) and 272 (iv and po), respectively on a qdx5 for two weeks schedule. For pharmacokinetic studies, the mice were administered all three analogues iv at 20 mg/kg and 272 was also given at 50 mg/kg iv and po. Plasma and tissues were collected between 5 and 1440 min. Concentrations of the analogues were determined by LC-MS/MS and non-compartmental analysis was performed using PK solutions. Results: The IC50's of 601, 599 and 272 against Daudi cells in vitro were 2.9 µM, 25.4 µM and 10.6 µM, respectively. The mean tumor time to two doublings for 601, 599 or 272 at 20 mg/kg iv were 16.5±0.9 , 12.7±3.3 and 14.5±3.8 days, respectively, compared with control (11.7±3.9 days). At 50 mg/kg, the mean tumor time to two doublings for 601(iv), or 272 (iv or po) were 10.7±6.0, 6.7±2.6 and 5.3±1.6 days, respectively, compared with control (7.1±2.9 days). Following a single iv dose with 20 mg/kg of the analogues or 50 mg/kg of 272, plasma and tumor Cmax occurred at 5 min. Plasma and tumor Cmax occurred at 30 min for 272 po. AUC0-t for 601, 599 and 272 at 20 mg/kg iv were 1219, 5417, and 9411 µg•min/ml, respectively. AUC0-t for 272 at 50 mg/kg iv and po were 21908 and 7184 µg•min/ml, respectively. Plasma t1/2 of 601, 599 and 272 at 20 mg/kg iv were approximately 165, 38, and 454 min respectively. Plasma t1/2 of 272 at 50 mg/kg iv or po were 763 and 239 min, respectively. Clearances for 601, 599 at 20 mg/kg iv and 272 at 50 mg/kg iv or po in plasma were 15.3, 3.7, 2.1, 2.5 and 7.0 ml/min/kg, respectively. Conclusion: 601 had the lowest IC50 against Daudi cells in vitro and resulted in a significant delay in tumor time to two doublings after 20 mg/kg iv. There were no significant effects of the other celastrol analogues on Daudi lymphoma growth in vivo at 20 mg/kg and 50 mg/kg iv or po. 601 is under further preclinical evaluation against other human tumor xenografts. Support: R01-CA078039 and P30-CA47904 Citation Format: Jianxia Guo, Kathleen Paul, Edward V. Prochownik, Nicholas Cosford, Robert A. Parise, Jan H. Beumer, Julie L. Eiseman. Efficacy, pharmacokinetics and tissue distribution of celastrol analogues SBI 064601, SBI 0640599 and SBI 0069272 in C.B-17 SCID mice bearing Daudi Burkitt's lymphoma xenografts. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4644. doi:10.1158/1538-7445.AM2014-4644

Immunornase Therapy Of Lymphoma With Novel CD22-Targeting Quadruple Ranpirnase

Ranpirnase (Rap) is an amphibian ribonuclease showing anti-tumor activity in clinical studies. We have previously reported that targeted delivery of Rap by chemical conjugation or recombinant fusion with antibodies specific for CD22, CD74 and Trop-2 could enhance its in vitro cytotoxicity as high as 10,000-fold in selected malignant cell lines. The DOCK-AND-LOCKTM (DNLTM) platform technology is a powerful method to construct novel agents of defined composition and retained bioactivity by site-specific conjugation of two types of modules, one containing the dimerization and docking domain (DDD) of cAMP-dependent protein kinase A (PKA), referred to as the DDD module, and the other bearing the anchoring domain (AD) of an interactive A-kinase anchoring protein (AKAP), referred to as the AD module. Among the distinctive features of DNL are the spontaneous formation of a dimer of the DDD module and the self-assembly of the DDD module with the AD module into a non-covalent complex, which is subsequently rendered covalent with disulfide bonds to enhance stability in vivo. The amino acid sequences of a pair of DDD and AD linkers useful for the DNL conjugation are termed DDD2 and AD2, respectively. To further explore the potential of Rap-based immunotoxins, we expressed a DDD2-module of Rap in E. coli and linked the resulting dimer of Rap to an AD2-module of a humanized IgG (expressed in myeloma cells) at each of the carboxyl termini of either the light chain (the CK-format) or the heavy chain (the CH3-format), thus producing a class of novel immunoRNases with quadruple Rap. To date, we have evaluated a pair of such constructs, 22* -Rap and 22-Rap, comprising four copies of Rap linked to the CK or CH3 termini of epratuzumab (humanized anti-CD22), respectively, in a panel of CD22-expressing human lymphoma/leukemia cell lines, which include Burkitt lymphoma (Daudi, Raji, Ramos), acute lymphoblastic leukemia (REH, 697, and RS4;11), and mantle cell lymphoma (Granta-519 and Jeko-1). The results of the MTS assay indicated that 22* -Rap was highly active (EC50 ≤ 1 nM) against Daudi, Ramos, Raji, REH, 697, RS4;11, and Granta-519 cells. Comparable cytotoxicity with EC50 values in the subnanomolar range also was observed for 22-Rap in Ramos, Daudi and Jeko-1 cells. In contrast, neither the individual DNL components (IgG-AD2 or Rap-DDD2), tested alone or in combination, nor E1* -Rap, the counterpart of 22* -Rap, generated by substituting epratuzumab with hRS7 IgG (humanized anti-Trop-2), showed notable cytotoxicity. In a disseminated Daudi xenograft model in which treatment with 10 or 20 µg of 22* -Rap (q4dx4) started 7 days after intravenous inoculation of the tumor cells, all 10 mice (5 in each group) survived over 126 days and were tumor-free, whereas the control groups, treated with saline, epratuzumab (25 µg, q4dx4), or the same dose-schedule of nonspecific control, E1* -Rap, succumbed within 36 days. These promising results, together with the findings that 22* -Rap at 10 nM was marginally toxic to B cells and other hematological cells in PBMCs, encourage further development of 22* -Rap or 22-Rap for therapy of CD22-expressing lymphomas and leukemias. Disclosures:Liu:IBC Pharmaceuticals, Inc.: Employment, Stock option, Stock option Other; Immunomedics, Inc.: Employment, Stock option Other. Cardillo:Immunomedics, Inc.: Employment, Stock option Other. Goldenberg:Immunomedics: Employment, stock options, stock options Patents & Royalties. Chang:IBC Pharmaceuticals, Inc.: Employment, Stock option, Stock option Other; Immunomedics, Inc: Employment, Stock option Other.

Abstract 1019: Cytotoxicity in vitro, pharmacokinetics and tissue distribution of Link-N3 and Link-F3, two bivalent small molecules inhibitors of c-Myc/Max interactions in C.B-17 SCID mice bearing Daudi Burkitt's lymphoma xenografts.

Abstract Introduction: c-Myc plays an important role in cellular proliferation, differentiation, apoptosis and cell cycle progression and its overexpression is associated with aggressiveness and poor prognosis in many cancers. In order to be transcriptionally active, c-Myc must heterodimerize via its bHLH-ZIP domain with its obligatory partner Max. Two small molecules that prevent c-Myc/Max heterodimerization and inhibit c-Myc function are 10074-G5 and 10058-F4. Analogues of these small molecule c-Myc inhibitors have been linked together by a flexible bridge to form Link-N3 and Link-F3. We have characterized the ability of these Link analogues to inhibit the in vitro growth of a c-Myc-overexpressing human cell line, Daudi Burkitt's lymphoma. We evaluated their pharmacokinetics, metabolism and efficacy in mice bearing Daudi xenografts. Methods: Inhibition of Daudi cell growth by Link-N3 and Link-F3 was assessed by MTT assay after 72 h of incubation. Accumulation of Link-N3 and Link-F3 by Daudi cells incubated with 10 μM of Link-N3 and Link-F3 was measured by HPLC. For pharmacokinetic and metabolic studies, C.B-17 SCID mice bearing Daudi xenografts were treated with 10 mg/kg Link-N3 or 5mg/kg Link-F3 IV. Plasma and tissues were collected between 5 and 1440 min. Urine was collected at 360 and 1440 min. Concentrations of Link-N3 and Link-F3 were determined by HPLC-UV, and metabolites were identified by LC-MS/MS. For efficacy compounds were administered qdx4 at the same doses. Results: The IC50’s for Link-N3 and Link-F3 against Daudi cells could not be determined because compounds precipitated at concentrations above 30 μM. Daudi cells accumulated Link-N3 and Link-F3 ∼19-fold and ∼17-fold, respectively, with peak intracellular concentrations between 3 and 6 h of incubation. Neither compound resulted in tumor growth inhibition following IV dosing. Following a single IV dose of 10 mg/kg Link-N3 or 5 mg/kg Link-F3, peak plasma concentrations at 5 min were 27.3 μg/ml and 5.3 μg/ml, respectively. Plasma Auc0-t for Link-N3 and Link-F3 were 1759.6 μg·min/ml and 137.9 μg·min/ml, respectively. Plasma T1/2 of Link-N3 and Link-F3 were 394 and 7 min, respectively. The volume of distribution was approximately 3204 ml/kg and 35 ml/kg for Link-N3 and Link-F3, respectively. Clearances for Link-N3 and Link-F3 were 5.6 ml/min/kg and 3.6 ml/min/kg, respectively. Peak tumor concentration of Link-N3 at 30 min was 0.77 μg/g and Link-F3 at 5 min was 0.49 μg/g, both much lower than peak plasma concentrations. Conclusion: The lack of significant antitumor activity of Link-N3 or Link-F3 in tumor-bearing mice is related to the low concentrations that reached the xenografts. Modification will be required in these bivalent compounds to make them more drug-like. Support: 1R01CA142580 and P30-CA47904 Citation Format: Jianxia Guo, Dana M. Clausen, Edward V. Prochownik, Steven J. Metallo, Robert A. Parise, Jan H. Beumer, Julie L. Eiseman. Cytotoxicity in vitro, pharmacokinetics and tissue distribution of Link-N3 and Link-F3, two bivalent small molecules inhibitors of c-Myc/Max interactions in C.B-17 SCID mice bearing Daudi Burkitt's lymphoma xenografts. [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 1019. doi:10.1158/1538-7445.AM2013-1019

Abstract 1803: HLA-DR-targeting tetrameric IFNα2b immunocytokine has potent in vitro and in vivo activity in myelomas, lymphomas and leukemias

Abstract Introduction: IFNα2 has been used in therapy of a variety of hematopoietic neoplasias, including myeloma (MM), NHL, hairy cell leukemia (HCL), and CML. However, its therapeutic potential is limited by its short circulating half-life and systemic toxicity. Fusion of IFNα2 to a monoclonal antibody (mAb) improves solubility and stability, and markedly increases circulating half-life. In addition to allowing less frequent and lower doses via extended Pk, targeting of IFNα2 to tumor sites using a tumor-directed mAb can reduce systemic concentrations and increase local concentration and tumor retention of IFNα2, thereby improving the therapeutic index. Increased tumor concentrations of IFNα2 can augment its direct anti-proliferative, apoptotic and anti-angiogenic activity, as well as prime an antitumor immune response. Methods: We previously used the DNL method to generate stably-tethered immunocytokines (Rossi et al., Blood 2009;114:3864-71; Rossi et al., Cancer Res 2010;70:7600-9), which include 20-2b-2b (CD20-targeted mAb-IFNα comprising tetrameric IFNα2b and veltuzumab) and 20-C2-2b (bispecific CD20/HLA-DR-targeting dimeric IFNα2b). Here we describe the potent in vitro and in vivo anti-tumor activities of a third immunocytokine, C2-2b-2b, comprising tetrameric IFNα2b and the anti-HLA-DR mAb, hL243, against myeloma, lymphoma and leukemia cell lines. Results: C2-2b-2b bound target cells with similar avidity to hL243 and exhibited high IFNα specific activity. In vitro, C2-2b-2b inhibited a panel of 20 cell lines comprising NHL (Burkitt, mantle cell & follicular), leukemia (HCL, AML, ALL & CLL), and MM, and in most cases was more effective than CD20-targeted mAb-IFNα or a mixture comprising hL243 and IFNα. Our findings indicate that a given cell's responsiveness depends on HLA-DR expression/density and its sensitivity to IFNα and hL243. C2-2b-2b was remarkably potent in vivo, where a single 1 μg dose significantly improved survival in advanced Daudi (NHL) and CAG (MM) xenograft models, and doses of ≥10 μg resulted in 70-100% long-term survivors (cures). C2-2b-2b showed superior anti-tumor efficacy compared to hL243 IgG, hL243 + rIFNα2b, Peginterferonalfa-2a or non-targeting mAb-IFNα. Ex-vivo pharmacodynamics studies using whole blood spiked with either NHL or MM cells showed that C2-2b-2b depleted either tumor cell type more efficiently than normal B cells and monocytes, did not deplete T cells, and was cytotoxic to dendritic cells, with myeloid DCs more susceptible than plasmacytoid DCs. Conclusions: The DNL method provides a modular approach to efficiently tether multimeric cytokines onto a targeting antibody, resulting in higher in vivo potency than the original cytokines due to improved pharmacokinetics, targeting and reduced systemic toxicity. These results suggest that C2-2b-2b might be useful in the treatment of various hematopoietic malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1803. doi:10.1158/1538-7445.AM2011-1803

In Vitro Cytotoxicity and In Vivo Efficacy, Pharmacokinetics, and Metabolism of 10074-G5, a Novel Small-Molecule Inhibitor of c-Myc/Max Dimerization

The c-Myc oncoprotein is overexpressed in many tumors and is essential for maintaining the proliferation of transformed cells. To function as a transcription factor, c-Myc must dimerize with Max via the basic helix-loop-helix leucine zipper protein (bHLH-ZIP) domains in each protein. The small molecule 7-nitro-N-(2-phenylphenyl)-2,1,3-benzoxadiazol-4-amine (10074-G5) binds to and distorts the bHLH-ZIP domain of c-Myc, thereby inhibiting c-Myc/Max heterodimer formation and inhibiting its transcriptional activity. We report in vitro cytotoxicity and in vivo efficacy, pharmacodynamics, pharmacokinetics, and metabolism of 10074-G5 in human xenograft-bearing mice. In vitro, 10074-G5 inhibited the growth of Daudi Burkitt's lymphoma cells and disrupted c-Myc/Max dimerization. 10074-G5 had no effect on the growth of Daudi xenografts in C.B-17 SCID mice that were treated with 20 mg/kg 10074-G5 intravenously for 5 consecutive days. Inhibition of c-Myc/Max dimerization in Daudi xenografts was not seen 2 or 24 h after treatment. Concentrations of 10074-G5 in various matrices were determined by high-performance liquid chromatography-UV, and metabolites of 10074-G5 were identified by liquid chromatography/tandem mass spectrometry. The plasma half-life of 10074-G5 in mice treated with 20 mg/kg i.v. was 37 min, and peak plasma concentration was 58 μM, which was 10-fold higher than peak tumor concentration. The lack of antitumor activity probably was caused by the rapid metabolism of 10074-G5 to inactive metabolites, resulting in tumor concentrations of 10074-G5 insufficient to inhibit c-Myc/Max dimerization. Our identification of 10074-G5 metabolites in mice will help design new, more metabolically stable small-molecule inhibitors of c-Myc.

Abstract 2452: Efficacy and toxicity of an anti-CD19 antibody drug conjugate

Abstract CD19 is an attractive target for immunotherapy. The expression of CD19 is restricted to B cells and follicular dendritic cells. In B cells, expression is initiated at the point of B lineage commitment and continues through the pre B and mature B cell differentiation. Expression is lost during the terminal differentiation of B cells to plasma cells. CD19 expression continues in B-lineage cells that have undergone neoplastic transformation, making it a useful marker in diagnosis and treatment of B cell derived lymphomas and leukemias, including non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL) and chronic lymphocytic leukemia (CLL). We have generated a fully human antibody to CD19 (MDX-1435) in transgenic mice for high binding affinity as well as the ability to rapidly internalize for drug delivery. The antibody was conjugated to a potent DNA alkylating agent related to Duocarmycin (MED-2460). This ADC (MDX-1206) shows anti-tumor activity in established Raji, Daudi and Ramos subcutaneous xenograft models at doses as low as 0.1 μmole/kg with no associated weight loss, or other overt toxicity. Efficacy was substantially superior to that of MDX-1435 and non-specific control antibody conjugates. In addition, MDX-1435 binds to cynomologus monkey CD19 with similar affinity and tissue distribution as in humans. The intravenous administration of two doses at 0.4 μmole/kg was well tolerated in cynomologus monkeys and did not result in overt clinical or histological signs of toxicity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2452.