Number of complete responders (CRs) are indicated in the physique. TICAs were evaluated in a suite of and assays to characterize their pharmacology and mechanism of action. Results Linking against costimulatory receptors (e.g., CD137) to against tumor antigens (e.g., EphA2) created potent agonists that activated the receptors selectively in the presence of tumor cells expressing these antigens. An EphA2/CD137 TICA (“type”:”entrez-protein”,”attrs”:”text”:”BCY12491″,”term_id”:”2055125703″,”term_text”:”BCY12491″BCY12491) efficiently costimulated human peripheral blood mononuclear cells in the presence of Cefuroxime sodium EphA2 expressing tumor cell lines as measured by the increased secretion of interferon and interleukin-2. Treatment of C57/Bl6 mice transgenic for the human CD137 extracellular domain name (huCD137) bearing EphA2-expressing MC38 tumors with “type”:”entrez-protein”,”attrs”:”text”:”BCY12491″,”term_id”:”2055125703″,”term_text”:”BCY12491″BCY12491 resulted in the infiltration of CD8+ T cells, elimination of tumors and generation of immunological memory. “type”:”entrez-protein”,”attrs”:”text”:”BCY12491″,”term_id”:”2055125703″,”term_text”:”BCY12491″BCY12491 was cleared quickly from the circulation (plasma t1/2 in mice of 1C2 hr), yet intermittent dosing proved effective. Conclusion Tumor target-dependent CD137 agonism using a novel chemical approach (TICAs) afforded elimination of tumors with only intermittent dosing suggesting potential for a wide therapeutic index in humans. This work unlocks a new path to effective cancer immunotherapy via agonism of TNF superfamily receptors. are ideally positioned to address these challenges in a way not practically feasible with traditional antibodies2 3 9 11 or bispecific biological agents.12C14 offer small highly modular building blocks, 15 16 rapid tissue distribution and penetration,17 rapid clearance compared with biologics16 18 and drug-like characteristics.18 19 Methods Cell lines and reagents HT-1376, NCI-H292, PC-3, RKO, A549, LNCaP, HT-29, HT-1080, CT26 and 4T1 cells were obtained from ATCC. MC38 cells were obtained from the National Cancer Institute (L-159-2018/1). 4T1 cells were engineered to express mouse Nectin-4 (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_027893.3″,”term_id”:”170014681″,”term_text”:”NM_027893.3″NM_027893.3) using CRISPR/Cas9 gene editing. In brief, a targeting vector carrying the mouse Nectin-4 coding sequence and homology arms recognizing the Rosa26 locus was transiently transfected into 4T1 cells along with plasmids expressing the appropriate sgRNAs. Single cell clones were isolated, and mouse Nectin-4 levels were evaluated by flow cytometry. Nectin-4 overexpressing MC38 and CT26 cell lines were generated PF4 by lentiviral contamination using pLenti6.3-CMV-MCS vector (Invitrogen) with Nectin4 CDS region (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_027893.3″,”term_id”:”170014681″,”term_text”:”NM_027893.3″NM_027893.3) at MCS (AscI and PmeI). Virus was prepared by transfecting 293FT cells (Invitrogen) with psPAX, VSV-G and Nectin-4 (or NT) vectors. CT26 or MC38 cells were transduced by Nectin-4 or NT lentivirus and selected with Blasticidin. Several individual clones were screened by flow cytometry (anti-Nectin-4, AF2659, R&D Systems) to select clones with appropriate level of Nectin-4 expression. Clones CT26#7 and MC38#13 were selected for further studies. Nectin-4 overexpression in CT26#7 and MC38#13 tumor tissues was verified by flow cytometry from single cell suspensions prepared from tumors grown in syngeneic Balb/c (CT26#7) or C57Bl/6 (MC38#13) mice. Human peripheral blood mononuclear cells (PBMCs) from healthy volunteer donors were isolated from buffy coats (Oklahoma Blood Institute, Oklahoma City, Oklahoma, USA) by ficoll centrifugation gradient (GE Healthcare Ficoll-Paque PLUS Media), followed by red blood cell lysis with ACK lysing buffer (Gibco). Anti-CD137 mAbs: Urelumab was prepared by Biocytogen (Beijing, China) using ExpiCHO expression system via transient transfection using sequence from published patent (US8137667B2) and purified by protein A affinity chromatography or purchased from Creative Biolabs (TAB-179). Utomilumab was prepared using HEK293 expression system via transient transfection using heavy chain (LC) and light chain (HC) regions from published patent (US20120237498A1) and purified using protein G affinity chromatography. Recombinant proteins: The following proteins were purchased for use in SPR experiments or reporter cell assays. Human proteins: CD137 Cefuroxime sodium (92204B, R&D Systems), CD137L (2295-4L, R&D Systems), OX40-Fc (OXO-H5255, Acro Biosystems), OX40 (OX0-H5224, Acro Biosystems), OX40L (OXL-H52Q8, Acro Biosystems), CD40 (CD40-H5228, Acro Biosystems), programmed death ligand 1 (PD-L1) (9049-B7, R&D Systems), EphA1-Fc (15789-H02H, Sino Biologics), EphA3-Fc (6444-A3, R&D Systems), EphA4-Fc (11314-H03H, Sino Biologics), EphA5 Cefuroxime sodium (3036-A5, R&D Systems), EphA6-Fc (5606-A6, R&D Systems), EphB4-Fc (10235-H02H, Sino Biologics), Nectin-1 (2880-N1, R&D Systems), Nectin-2 (2229-N2, R&D Systems), Nectin-3 (3064-N3, R&D Systems), Nectin-like-1 (3678-S4-050, R&D Systems), Nectin-like-2 (3519-S4-050, R&D Systems), Nectin-like-3 (4290-S4-050, R&D Systems), Nectin-like-4 (4164-S4, R&D Systems) and Nectin-like-5 (2530-CD-050, R&D Systems). Mouse proteins: CD137 (41B-M52H7, Acro Biosystems), EphA2 Cefuroxime sodium (50586-M08H, Sino Biological) Nectin-4 (3116-N4, R&D Systems) and PD-L1 (9048-B7-100, R&D Systems). The following proteins were expressed for use in SPR experiments. Human EphA2: The ecto domain name of human EphA2 (Lys27-Asn529) was cloned into pEXPR-IBA44, between the 5 NheI and 3 BsaI sites. Protein was expressed in HEK293 cells. It was purified by IMAC and gel filtration chromatography. Human Nectin-4 was prepared at Charles River Labs. Human Nectin-4 (residues Gly32-Ser349; NCBI RefSeq: “type”:”entrez-protein”,”attrs”:”text”:”NP_112178.2″,”term_id”:”222136611″,”term_text”:”NP_112178.2″NP_112178.2) with a gp67 signal sequence and C-terminal FLAG tag was cloned into pFastbac-1 and baculovirus made using standard Bac-to-Bac protocols (Life Technologies). Sf21 cells at 1 x 106/mL in Excell-420 medium (Sigma) at 27 C were infected at a MOI of 2 with a P1 virus stock and the supernatant harvested at 72 hr. The supernatant was batch bound.
Number of complete responders (CRs) are indicated in the physique
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a 67 kDa type I transmembrane glycoprotein present on myeloid progenitors
and differentiation. The protein kinase family is one of the largest families of proteins in eukaryotes
Apoptosis
bladder
brain
breast
cell cycle progression
cervix
CSP-B
Cyproterone acetate
EGFR) is the prototype member of the type 1 receptor tyrosine kinases. EGFR overexpression in tumors indicates poor prognosis and is observed in tumors of the head and neck
EM9
endometrium
erythrocytes
F3
Goat polyclonal to IgG H+L)
Goat polyclonal to IgG H+L)Biotin)
GRK4
GSK1904529A
Igf1
Mapkap1
monocytes andgranulocytes. CD33 is absent on lymphocytes
Mouse monoclonal to CD33.CT65 reacts with CD33 andtigen
Palomid 529
platelets
PTK) or serine/threonine
Rabbit Polyclonal to ARNT.
Rabbit polyclonal to BMPR2
Rabbit Polyclonal to CCBP2.
Rabbit Polyclonal to EDG4
Rabbit polyclonal to EIF4E.
Rabbit polyclonal to IL11RA
Rabbit polyclonal to LRRIQ3
Rabbit Polyclonal to MCM3 phospho-Thr722)
Rabbit Polyclonal to RBM34
SB 216763
SKI-606
SNX-5422
STK) kinase catalytic domains. Epidermal Growth factor receptor
stomach
stomach and in squamous cell carcinoma.
TNFSF8
TSHR
VEGFA
vulva