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)

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

Pang HW, Linares A, Couling L, Santollo J, Ancheta L, Daniels D, Speth RC (2019) Novel high molecular weight albumin-conjugated angiotensin II activates β-arrestin and G-protein pathways. Endocrine 66(2):349-359. doi: 10.1007/s12020-019-01930-z

Objective: To study the ability of a novel bovine serum albumin-angiotensin II (BSA-Ang II) conjugate to effect responses of the AT1 angiotensin II receptor subtype mediated by the G-protein-coupled and the beta-arrestin pathways.

Summary: BSA-Ang II and Ang II stimulated water appetite equivalently but BSA-Ang II stimulated saline appetite more than Ang II. Both BSA-Ang II and Ang II were considerably more potent at causing calcium mobilization than β-arrestin binding.

Usage: Angiotensin II (Ang II) was conjugated with bovine serum albumin and compared with Ang II for competition binding to AT1 receptors, to stimulate aldosterone release from adrenocortical cells, to promote beta-arrestin binding to AT1 receptors, to promote calcium mobilization, and stimulate drinking of water and saline by rats.

Related Products: Custom Conjugates

Goodman RL, Lopez JA, Bedenbaugh MN, Connors JM, Hardy SL< Hileman SM, Coolen LM, Lehman MN (2018) Evidence that the LH surge in ewes involves both neurokinin B-dependent and -independent actions of kisspeptin. Neuroscience 2018 Abstracts 773.20 / YY14. Society for Neuroscience, San Diego, CA.

Summary: It is generally recognized that kisspeptin plays a key role in induction of the LH surge in sheep and we have reported evidence that neurokinin B (NKB) does so as well. Specifically, disrupting NKB signaling in the retrochiasmatic area (RCh) using either an antagonist to its receptor, NK3R, or lesions of NK3R-containing neurons in the RCh with a saporin conjugate (NK3-SAP) reduced the amplitude of the estrogen-induced LH surge by 50%. Because a KISS1R antagonist (p271) also produced a 50% decrease in surge amplitude, we hypothesized that these two systems are organized in series with NKB actions in the RCh stimulating kisspeptin release. If this is the case, then the combination of NK3R lesions and a KISS1R antagonist should produce the same inhibition as either treatment alone. This experiment tested this prediction using a 2 x 2 design. Breeding season ewes were ovariectomized and immediately given an estradiol (E) implant sc and two progesterone implants (CIDRs) intravaginally that produced luteal phase levels of these steroids. Ewes then received bilateral injections of either NK3-SAP (n=6) or Blank-SAP (n=5) into the RCh. Three weeks later, an artificial follicular phase was produced by inserting four 3 cm long E implants 24 hrs after CIDR removal and either saline or p271 was infused into the lateral ventricle for 16-24 hrs after E implantation; LH was monitored every 2-4 hrs for two days. CIDRs were then reinserted and the protocol repeated in a cross-over design so that all ewes received saline and p271 treatment. In Blank-SAP ewes, p271 decreased the peak of the LH surge from 61.2 ± 7.6 to 27.4 ± 4.6 ng/mL and delayed it 8 hrs (from 26.5 ± 0.5 to 34.1 ± 1.2 hrs post E implantation). The NK3-SAP injections alone decreased the peak of the LH surge to 29.7 ± 10.7 ng/mL compared to Blank-SAP, but the peak was not further inhibited by p271 in these NK3-SAP-treated ewes (24.4 ± 1.4 ng/mL). However, p271 delayed the peak of the LH surge (from 28.8 ± 1.2 to 34.8 ± 2.1 hrs post E implantation) in the ewes injected with NK3-SAP. Based on these results, we propose that kisspeptin has two roles in the LH surge in ewes: it initiates the surge independent of NKB signaling in the RCh, and maintains LH secretion during the surge by a NKB-dependent system.

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