Non-Hodgkin lymphoma (NHL) affects over 400,000 people in the United States; its incidence increases with age. Treatment options are numerous and expanding, yet efficacy is often limited by toxicity, ...particularly in the elderly. Nearly 70% patients eventually die of the disease. Many patients explore less toxic alternative therapeutics proposed to boost anti-tumor immunity, despite a paucity of rigorous scientific data. Here we evaluate the lymphomacidal and immunomodulatory activities of a protein fraction isolated from fermented wheat germ. Fermented wheat germ extract was produced by fermenting wheat germ with Saccharomyces cerevisiae. A protein fraction was tested for lymphomacidal activity in vitro using NHL cell lines and in vivo using mouse xenografts. Mechanisms of action were explored in vitro by evaluating apoptosis and cell cycle and in vivo by immunophenotyping and measurement of NK cell activity. Potent lymphomacidal activity was observed in a panel of NHL cell lines and mice bearing NHL xenografts. This activity was not dependent on wheat germ agglutinin or benzoquinones. Fermented wheat germ proteins induced apoptosis in NHL cells, and augmented immune effector mechanisms, as measured by NK cell killing activity, degranulation and production of IFNγ. Fermented wheat germ extract can be easily produced and is efficacious in a human lymphoma xenograft model. The protein fraction is quantifiable and more potent, shows direct pro-apoptotic properties, and enhances immune-mediated tumor eradication. The results presented herein support the novel concept that proteins in fermented wheat germ have direct pro-apoptotic activity on lymphoma cells and augment host immune effector mechanisms.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Antibody drug conjugates (ADCs) can deliver potent drugs to cancer cells by employing the specificity of monoclonal antibodies (mAbs). ADCs have demonstrated significant anticancer activity and, in ...2011, brentuximab vedotin has been approved by the FDA for the treatment of Hodgkin's and anaplastic large cell lymphomas. CD22 is an ideal target for ADC against B-cell malignancies because of its lineage-specific expression and rapid internalization upon antibody binding. In this study, we evaluated the anti-CD22 mAb HB22.7 as a vehicle for the targeted delivery of the potent toxin saporin (SAP). In vitro, HB22.7-SAP was cytotoxic against a panel of non-Hodgkin's lymphoma (NHL) cell lines representing the most common types of the disease. Moreover, in a xenograft model of NHL, HB22.7-SAP significantly inhibited the growth of established lesions and completely prevented tumor development when treatment was initiated within 24 h from tumor-cell inoculation. HB22.7-SAP had no significant in vivo toxicity. In conclusion, HB22.7 constitutes a potential platform for CD22-targeted ADCs.
FWGP activates the immune system (TUM7P.930) Abuhay, Mastewal; O'Donnell, Robert; Ma, Yunpeng ...
The Journal of immunology (1950),
05/2014, Volume:
192, Issue:
1_Supplement
Journal Article
Peer reviewed
Abstract
The immune system is an essential defense against malignancy; immunosuppression increases the risk of developing cancer. A better understanding of how to modulate the immune system may lead ...to new methods to prevent and treat disease. While recent developments in targeted immunomodulation have improved cancer treatments, most therapeutics are toxic, expensive, or have marginal efficacy. We developed a natural product derived from fermented wheat germ extract (FWGE) that acts as an immune modulator. Through further purification we identified protein components, (fermented wheat germ proteins, FWGP), that are more potent than FWGE. In vitro, FWGP was cytotoxic against B cancer cells with IC50s ranging from 10μg/ml to 17.5μg/ml. In vivo studies showed substantial efficacy without toxicity. We found that FWGP was a potent immune modulator. It increased CD4+ T cells, NK-T cells, & activated monocytes by 4, 10, & 50 folds, respectively, while decreasing CD8+ T cells. CD56-bright NK cells treated with FWGP increased compared to the heat-inactivated FWGP treatment, and NK-mediated IFN-γ production rose as FWGP increased. Also, FWGP (0.1 mg/ml) activated complement with 2-fold potency compared to either control or FWGE (55mg/ml). It enhanced ADCC as well; PBMCs treated with FWGP showed a dose dependent increase in killing of Ramos. FWGP’s in vivo efficacy could be due to its immune modulating effects and may be the basis for a non-toxic treatment for lymphoma and other malignancies.
In this study, the HB22.7 anti-CD22 mAb, was used for specific, targeted delivery of the potent anti-cancer agent, monomethyl auristatin E (MMAE) to non-Hodgkin lymphoma (NHL). MMAE was covalently ...coupled to HB22.7 through a valine-citrulline peptide linker (vc). Maleimide-functionalized vcMMAE (mal-vcMMAE) was reacted with thiols of the partially reduced mAb. Approximately 4 molecules of MMAE were conjugated to HB22.7 as determined by residual thiol measurement and hydrophobic interaction chromatography-HPLC (HIC-HPLC). HB22.7-vcMMAE antibody drug conjugate (ADC) retained its binding to Ramos NHL cells and also exhibited potent and specific
in vitro
cytotoxicity on a panel of B cell NHL cell lines with IC
50
s of 20 - 284 ng/ml. HB22.7-vcMMAE also showed potent efficacy
in vivo
against established NHL xenografts using the DoHH2 and Granta 519 cell lines. One dose of the ADC induced complete and persistent response in all DoHH2 xenografts and 90% of Granta xenografts. Minimal toxicity was observed. In summary, HB22.7-vcMMAE is an effective ADC that should be evaluated for clinical translation.
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EMUNI, FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UL, UM, UPUK, VKSCE, ZAGLJ
Abstract
Background: Trophoblast antigen 2 (Trop2) or Tumor-Associated Calcium Signal Transducer 2 (TACSTD2) is a transmembrane glycoprotein that is overexpressed in several solid tumor cancers. ...Antibody drug conjugate (ADC) targeting of Trop2 has been clinically validated. The Trop2 ADC Trodelvy® (also known as Sacituzumab govitecan/IMMU-132) is approved for therapy of TNBC and urothelial cancer, and Datopotamab DXd (DS-1062) is currently being tested in NSCLC.
Peak Bio’s Thailanstatin suite of immunomodulatory linker-toxins (L-Ts) is a set of 7 related molecules with distinct ADC features that have been extensively characterized in vitro and in vivo as Her2 ADCs and is being used as a platform to generate a pipeline of potentially differentiated ADCs (AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1832).
Results: M2.8 is a fully humanized Peak Bio antibody (Ab) that targets human Trop2 (hTrop2) with high binding affinity Kd (M)= 8.97-09. M2.8 was optimized for binding affinity and humanization relative to its parent M2.1 Kd (M)= 1.78-08, and, both M2.1 and M2.8, display species-specificity for the human and cynomolgus monkey Trop2 proteins. When bound to hTrop2-positive cell lines, M2.1 and M2.8 can rapidly internalize and deliver an ADC payload.
To identify the optimal release for therapeutic efficacy, we conjugated our family of L-Ts with hRS7 at a comparable drug-to-antibody ratio (DAR) of 4. Maximal tumor growth inhibition (TGI) was associated with the Trop2 ADCs that were conjugated via amine coupling to lysine residues using non-cleavable L-T L22, later renamed PH1.
To determine the effect of antibody on TGI efficacy, different doses, and DARs of M2.1 PH1 ADC were compared to PH1 ADCs made from other Trop2 Abs. At low dose levels, M2.1 PH1 ADCs exhibited comparable TGI with hRS7 PH1, TINA PH1, and T6-16 PH1 ADCs, but exhibited significantly greater TGI than hRS7 SN38 (IMMU-132), suggesting that payload potency, rather than epitope, was important in targeting Trop2. The results also suggested that a PH1 DAR of 4 was sufficient to obtain 90% TGI with multiple anti-Trop2 Abs.
M2.1 PH1 ADC was safely tolerated by cynomolgus monkeys at 3 and 6 mg/kg Q3W x 3 doses with mild-to-moderate changes in liver enzymes and platelets that reversed to baseline within 7-10 days of administration of each dose.
The optimized M2.8 PH1 DAR4 ADCs killed Trop2-positive cell lines with single-digit nanomolar potency, while exhibiting low off-target cytotoxicity vs normal human skin fibroblasts and Trop2-negative cell lines in vitro. M2.8 PH1 ADC regressed all 200mm3 sized NCI-N87 tumors and 50% of these tumors exhibited long-term regression for 5 months.
Conclusion: The target validation studies suggest that Trop2 PH1 ADCs were sufficiently differentiated in areas of preclinical efficacy and well tolerated in a toxicologically relevant host.
Citation Format: Satyajit K. Mitra, William Monteith, Mary Do, Greg Tuffy, Scott Savage, Jeffrey Kang, Mastewal Abuhay, Teodora Losic, Sanjeevani Ghone, William E. Haskins, Vasu Jammalamadaka, Sanjeev Satyal. Rationale for the development of a differentiated Trop2 ADC 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 6297.
The prevalence of cancer continues to rise worldwideCancer is a leading cause of deaths worldwide. It is estimated about 1.7 million people, in the United States alone, will be diagnosed with cancer ...and about 0.6 million will die due to cancer, about a third of which are due to lung cancer and non-Hodgkin lymphoma (NHL) (1). The treatment options for lung cancer are improving but still inadequate and toxic (2, 3). The treatment options for NHL can frequently be effective but relapse due to resistance is common (4). In addition, efficacy of conventional cancer treatments, including those for lung cancer and NHL, are limited by systemic toxicities as most of these treatments are not targeted specifically to cancer cells. Therefore, developments of more selective therapeutics that target specific markers associated with malignancies may improve the response rates while minimizing systemic toxicity.To this end, as this thesis will discuss, we developed antibody-drug conjugate (ADC) and antibody-micelle conjugate delivery systems that selectively target CD22. CD22 is a surface marker that is expressed on most B-cell NHL, lung cancer, and possibly, other malignancies. As such, these delivery systems could be beneficial for treating several types of malignancies. Selective delivery of treatments into CD22+ cancer cells were formulated using the α-CD22 monoclonal antibodies (mAbs), HB22.7 and JT22.1. HB22.7 was conjugated with monomethyl auristatin E (MMAE) for active cellular targeting and delivery of the cytotoxic agent into CD22+ cells. In other studies, JT22.1 was conjugated to disulfide cross-linked micelles (DCM) for a combination of, both, active cellular targeting and passive tissue targeting.Active cellular targeted delivery systems are formulated with homing ligands such as mAbs to selectively deliver bound treatments by interacting with overexpressed surface molecules on target cells (5, 6). HB22.7 and JT22.1 are efficient homing ligands that selectively interact with CD22 and facilitate the internalization of their cargo through receptor mediated endocytosis. Thus, the antibodies and the target, CD22, are ideal for formulating targeted therapeutics that aim to selectively eradicate CD22+ cancer cells.Passive tissue targeting systems are formulated with nanocarriers to selectively deliver encapsulated treatments to cancer tissues via the enhanced permeability and retention (EPR) effect. Nanocarriers (NCs) passively extravasate and accumulate in tumor tissues due to leaky blood vessels and low-pressure lymphatic systems that are associated with cancer and other diseases (a phenomenon known as the EPR effect) (5-7). NCs such as DCM have high potential for enhancing the efficacy and safety profiles of conventional and new therapeutic agents by passively accumulating in tumor areas.The active targeting ADC formulation, HB22.7-vcMMAE, selectively eradicated CD22+ NHL cells lines in vitro and in vivo. The potent anti-microtubule agent, MMAE, was conjugated to HB22.7 using a valine – citrulline (vc) peptide linker. The protease sensitive peptide linker facilitates selective release of MMAE within target cells by stabilizing the ADC to minimize premature release of the cytotoxic agent while in the circulation, which improves efficacy and lowers systemic toxicity. Just one dose (7.5 mg/kg) of the ADC treatment resulted in complete responses in NHL (DoHH2 100% CR and Granta-519 90% CR) murine xenograft models. Significant toxicities were not observed. High response rates with just one dose, with minimal toxicity prompted further (clinical) investigations of HB22.7-vcMMAE and other α-CD22 mediated targeted formulations. The developments and treatments of HB22.7-vcMMAE are discussed in Chapter 2.The successes of α-CD22 mediated formulations, HB22.1-vcMMAE and HB22.7-SAP, stimulated the development of new generations of CD22 targeting therapeutics. Therefore, to capitalize on the efficient delivery potentials of α-CD22 mAbs and to formulate even more effective targeted therapeutics by combining active and passive delivery approaches, we developed multifunctional CD22-targeted micelle formulations. Antibody-micelle conjugates were formulated using the α-CD22 mAb, JT22.1, and drug-encapsulating disulfide-crosslinked micelles (DCM). Conceptually, these JT22.1 coated multifunctional micelles passively accumulate in tumor tissues where they can actively bind to and internalize CD22+ cancer cells. The physiochemical properties of these micelles have been optimized to increase their circulation retention time, penetration, and to selectively release their cargo upon exposure to intra-tumor and intracellular reducing conditions. JT22.1-DCM was conjugated using copper-free click chemistry to produce stable and biocompatible formulations. Using the specificity of the anti-CD22 antibody, JT22.1, we developed effective, targeted micellar formulations that may have higher efficacy and safety profiles than their conventional counterparts for the treatment of NHL, lung cancer, and other CD22+ malignancies. However, optimization of the homing molecules, conjugation method, the linker, and the nanocarrier was warranted. The development of JT22.1-micelles is further discussed in chapter 3.In addition to CD22 targeting therapeutics, this thesis also explores the development of fermented wheat germ proteins (FWGP) as a non-toxic cancertherapeutic. The experiments demonstrated that FWGP selectively kill cancer cells, synergistically enhance chemotherapeutics, and modulates the immune system. FWGP enhances the efficacy of currently used chemotherapeutics and biological treatments by sensitizing cancer cells and modulating the immune system. FWGP can be produced in large scale, with consistency quality, and low cost. Additional advantage of FWGP is that it could be used to treat multiple types of cancers. The development of FWGP is discussed in chapter 4.HB22.7-vcMMAE, JT22.1-DCM, and FWGP have promising therapeutic potentials. These formulations selectively kill cancer cells with minimal toxicity against healthy cells. More studies to optimize these formulations could benefit cancer patients and help control the disease. Moreover, optimal combinations of these safe and effective formulations could enhance their therapeutic impact.
In this study, HB22.7, an anti-CD22 monoclonal antibody, was used for specific, targeted delivery of monomethyl auristatin E (MMAE) to non-Hodgkin lymphoma (NHL). MMAE was covalently coupled to ...HB22.7 through a valine–citrulline peptide linker (vc). Maleimide-functionalized vcMMAE (mal-vcMMAE) was reacted with thiols of the partially reduced mAb. Approximately 4 molecules of MMAE were conjugated to HB22.7 as determined by residual thiol measurement and hydrophobic interaction chromatography–HPLC (HIC-HPLC). HB22.7–vcMMAE antibody–drug conjugate (ADC) retained its binding to Ramos NHL cells and also exhibited potent and specific in vitro cytotoxicity on a panel of B cell NHL cell lines with IC
50
s of 20–284 ng/ml. HB22.7–vcMMAE also showed potent efficacy in vivo against established NHL xenografts using the DoHH2 and Granta 519 cell lines. One dose of the ADC induced complete and persistent response in all DoHH2 xenografts and 90 % of Granta xenografts. Minimal toxicity was observed. In summary, HB22.7–vcMMAE is an effective ADC that should be evaluated for clinical translation.
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EMUNI, FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UL, UM, UPUK, VKSCE, ZAGLJ
Abstract Targeted therapies, such as those using imatinib and rituximab, have revolutionized the treatment of Philadelphia chromosome-positive and CD20-positive acute lymphoblastic leukemia (ALL) ...respectively, yet these therapies are effective in only a subset of patients and remission is generally not durable. The next generation of targeted therapies includes the use of antibodies conjugated to potent cytotoxic agents and are classified as antibody drug conjugates (ADC). For B-lineage ALL, CD22 is an ideal target for ADC therapy because it is expressed on the majority of B-lineage ALL cells and because antibody binding mediates receptor internalization. HB22.7-SAP is a conjugate of our anti-CD22 monoclonal antibody (mAb), HB22.7, and the ribosome inhibiting protein, saporin (SAP). In vitro , HB22.7-SAP effectively bound to CD22 on the surface of pre-B ALL cell lines and exhibited potent and specific cytotoxicity. In a NOD/SCID xenograft mouse model of pre-B ALL, when compared to the vehicle-treated control, HB22.7-SAP increased the median survival time from 20 days to over 50 days without significant toxicity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Highlights • We propose that AR42 enhances HB22.7's efficacy by increasing CD22 surface levels. • HB22.7 and AR42 showed synergistic lymphomacidal activity in vitro and in vivo. • AR42 inhibited ...HB22.7-mediated CD22 internalization. • HB22.7 with HDAC inhibition may be a safe and efficacious NHL therapy.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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