custom-conjugates

Goodman RL, Moore AM, Onslow K, Hileman SM, Hardy SL, Bowdridge EC, Walters BA, Agus S, Griesgraber MJ, Aerts EG, Lehman MN, Coolen LM (2023) Lesions of kndy and kiss1r neurons in the arcuate nucleus produce different effects on lh pulse patterns in female sheep. Endocrinology 164(11):bqad148. doi: 10.1210/endocr/bqad148 PMID: 37776515

Objective: To test the functional role of ovine KNDy neurons in pulse generation and identify the roles of nearby Kiss1 receptor (Kiss1R)-containing cells.

Summary: Injection of NK3-SAP (NKB-SAP) ablated over 90% of the KNDy cells, Kiss-SAP lesioned about two-thirds of the Kiss1R population. This led to a significant decrease in LH pulse amplitude and altering LH pulse patterns. NK3-SAP increased the interpulse interval without affecting the regularity of LH pulses, whereas Kiss-SAP disrupted their regular hourly occurrence but not the interpulse interval. The findings suggest that KNDy neurons are critical for GnRH pulse generation in ewes, while ARC Kiss1R cells support the amplitude and regularity of these pulses, possibly as part of a positive feedback loop involving GABA or glutamate.

Usage: Saporin conjugates were injected into the arcuate nucleus. Kiss-SAP (kisspeptin54-SAP) was diluted to 700 ng/μL in PBS immediately before use. In preliminary work to test the effectiveness of Kiss-SAP, a single unilateral injection (1 μL of 700 ng/μL) of this conjugate was made in the preoptic area of 3 ewes. The contralateral side was used as control and either received no injections or Blank-SAP (1 μL of 700 ng/μL) (IT-21).

Related Products: NKB-SAP (Cat. #IT-63), Blank-SAP (Cat. #IT-21)

Marquez J (2023) PTGFRN as a target for antibody-drug conjugate (ADC) development in mesothelioma and medulloblastoma. Univ Maryland Baltimore Thesis.

Objective: To investigate the role of Prostaglandin F2 Receptor Negative (PTGFRN) regulator in cancer progression and develop an antibody-drug conjugate (ADC) targeting PTGFRN for the treatment of mesothelioma and pediatric medulloblastoma.

Summary: This dissertation explores the expression and function of PTGFRN in mesothelioma and pediatric medulloblastoma, identifying its association with aggressive cancer phenotypes. The study further develops a potent ADC using a PTGFRN-specific monoclonal antibody conjugated to the cytotoxic compound Duocarmycin, demonstrating significant anti-cancer efficacy in both in vitro and in vivo models .

Usage: Both a custom direct conjugate and Fab-ZAP Mouse (IT-48) were used (up to 10 nM) on transfected HEK-293A cells.

Related Products: Fab-ZAP mouse (Cat. #IT-48), Custom Conjugates

Le Z, Pan Q, He A, Liu H, Shi Y, Liu L, Liu Z, Ping Y, Chen Y (2023) Direct cytosolic delivery of proteins and CRISPR-Cas9 genome editing by gemini amphiphiles via non-endocytic translocation pathways. ACS Cent Sci 9(7):1313-1326. doi: 10.1021/acscentsci.3c00207 PMID: 37521791

Objective: Create a library composed of 150 gemini amphiphiles (GAs), a molecule with polar and nonpolar regions, to identify means to facilitate delivery of saporin across the cell membrane.

Summary: The gemini amphiphiles studied differ in the spacing of functional groups, hydrophobicity and sterics. A Cas9 ribonucleoprotein conjugated to saporin showed highest efficacy for internalization. HeLa cells were used to study the efffects of this found gemini-amphiphile conjugated to saporin.

Usage: A1I2-1R2C18 [a gemini ampiphile] conjugated to saporin and treated against HeLa cells at 0.0625, 0.125, 0.25, 0.5, 1μg/mL for 4 hours and then assessed for cell viability.

Q: We recently spoke to you about performing a custom saporin conjugation using our antibody. Is 0.09% azide in PBS in the antibody stock acceptable?

A: There are a number of dialysis steps within the conjugation protocol that will ultimately remove the azide from your antibody solution. So as long as your antibody will be happy in PBS without azide during the procedure, sending the material in 0.09% azide is fine. The final conjugate will be returned to you in PBS, sterile-filtered, without azide.

Q: In general, how many saporin molecules are incorporated per antibody? Can we test this by HPLC?

A: We aim for 2-2.5 moles of saporin per mole of antibody. You should be able to see differences in HPLC between your antibody with one vs. two vs. three saporins attached, however we will provide you with a saporin molar ratio and a product that has had free saporin and free antibody removed from the final conjugate.

Related: Custom Conjugates

Masoumi KC, Huang X, Sime W, Mirkov A, Munksgaard Thorén M, Massoumi R, Lundgren-Åkerlund E (2021) Integrin α10-antibodies reduce glioblastoma tumor growth and cell migration. Cancers (Basel) 13(5):1184. doi: 10.3390/cancers13051184

Summary: The authors investigated the treatment effect of two antibodies that have been developed to target the protein integrin 10, which is present on the surface of Glioblastoma (GB) cells. Function-blocking integrin alpha10, beta1-antibodies inhibit GB tumor growth as well as the migration of GB cells. This further validates integrin alpha10, beta1 as a promising target in GB and suggests a novel therapeutic strategy for the treatment of GB and other high-grade gliomas.

Usage: Infusions of anti-integrin α10β1-SAP or Anti-ctrl-SAP were made icv (1 µg/2 L per infusion).

See: Thorén MM et al. Integrin α10, a Novel Therapeutic Target in Glioblastoma, Regulates Cell Migration, Proliferation, and Survival. Cancers (Basel) 11(4):587, 2019.

Related Products: Custom Conjugates

Marquez J, Dong J, Dong C, Tian C, Serrero G (2021) Identification of prostaglandin F2 receptor negative regulator (PTGFRN) as an internalizable target in cancer cells for antibody-drug conjugate development. PLoS One 16(1):e0246197. doi: 10.1371/journal.pone.0246197

Summary: PTGFRN is a cell-surface protein that is upregulated in certain cancer types, including head and neck and, notably, pediatric medulloblastoma, an aggressive cancer with limited therapeutic options. With the selection of the mouse monoclonal antibody 33B7, the authors identified PTGFRN as a potential therapy target, and show that it is internalized by incubation with 33B7. Purified 33B7 antibody was sent to Advanced Targeting Systems where saporin was directly conjugated to the Fc region of 33B7 using their proprietary cleavable linker.

Usage: In a 96-well plate, 2000 cells/well were plated in triplicate in 100 μL of DMEM/F12 medium supplemented with 2.5% FBS, 0.4 ug/ml 33B7 antibody, and 0.9ug/ml of Fab-ZAP mouse. As an isotype control, cells were incubated with mouse Fab IgG-SAP as control (instead of 33B7) and Fab-ZAP.

Related Products: Fab-ZAP mouse (Cat. #IT-48), Fab IgG-SAP (Cat. #IT-67), Custom Conjugates

Knuplez E, Krier-Burris R, Cao Y, Marsche G, O’Sullivan J, Bochner BS (2020) Superior mouse eosinophil depletion in vivo targeting transgenic Siglec-8 instead of endogenous Siglec-F: Mechanisms and pitfalls. J Leukoc Biol 108:43-58. doi: 10.1002/jlb.3hi0120-381r

Objective: To determine if targeting Siglec-8 with mouse IgG1 antibodies, rather than targeting Siglec-F with rat IgG antibodies, in mice transgenic for Siglec-8, will prove to be a more effective strategy for eliminating mouse eosinophils in vivo.

Summary: This study is the first to describe a novel mouse strain of Siglec-8+F− eosinophils—a useful tool for studying human Siglec biology in preclinical models.

Usage: Siglec-8+F− mouse eosinophils were pretreated with 10 μg/mL saporin-conjugated isotype controls (mouse IgG1 or rat IgG2), anti-Siglec-8 (2C4) or anti-Siglec-F (9C7) antibodies for 24 h.

Related Products: Custom Conjugates

Qian L, Kasas L, Milne MR, Rawashdeh O, Marks N, Sharma A, Bellingham MC, Coulson EJ (2020) Cholinergic basal forebrain degeneration due to obstructive sleep apnoea increases Alzheimer’s pathology in mice. bioRxiv 2020.03.12.989848. doi: 10.1101/2020.03.12.989848

Usage: bilateral injections of urotensin II-saporin (UII-SAP; 0.07 μg/μL per site – unless stated otherwise; generous gift from Advanced Targeting Systems)

Related Products: Custom Conjugates, Blank-SAP (Cat. #IT-21)

Porter D, Moore A, Goodman R, Hileman S, Coolen L, Lehman M (2019) Cell-specific ablation of GnRH neurons using kisspeptin-saporin in the preoptic area of sheep, but not mice. J Endocrine Society 3(1):SAT–421. ENDO 2019 – 101st Annual Meeting of the Endocrine Society, New Orleans, Louisiana doi: 10.1210/js.2019-SAT-421

Objective: To invesigate the potential of integrin α10β1 as a therapeutic target in glioblastomas (GBMs).

Summary: Integrin α10β1 has a crucial role in the migration, proliferation, and survival of GBM cells and that an integrin α10β1 antibody–drug conjugate induced cell death of GBM cells both in vitro and in vivo.

Usage: Infusions of anti-10-SAP or Anti-ctrl-SAP were made icv (1 µg/2 L per infusion).

Related Products: Custom Conjugates

Thorén MM, Chmielarska Masoumi K, Krona C, Huang X, Kundu S, Schmidt L, Forsberg-Nilsson K, Floyd Keep M, Englund E, Nelander S, Holmqvist B, Lundgren-Åkerlund E (2019) Integrin α10, a novel therapeutic target in glioblastoma, regulates cell migration, proliferation, and survival. Cancers (Basel) 11(4):587. doi: 10.3390/cancers11040587 PMID: 31027305

Objective: To investigate the potential of integrin alpha10beta1 as a therapeutic target in glioblastomas (GBMs).

Summary: Integrin alpha10beta1 has a crucial role in the migration, proliferation, and survival of GBM cells and that an integrin alpha10beta1 antibody–drug conjugate induced cell death of GBM cells both in vitro and in vivo.

Usage: Infusions of anti- 10-SAP or Anti-ctrl-SAP were made icv (1 mcg/2 L per infusion).

Related Products: Custom Conjugates