Car t-cell therapy directed to lhr for the treatment of solid tumors
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example 1
n of Mouse Anti-LHR Monoclonal Antibodies
[0279]Antibodies against the lysine rich extracellular hormone binding domain of LHR were generated by repeated immunization of 4 week-old-BALB / c and NIH Swiss mice with genetically engineered LHR-Fc. As shown below in FIG. 3, the leader sequence and first part of the human LHR G-protein was used to generate the LHR-Fc used in the immunization and screening methods to generate and identify high binding antibodies. Since flow cytometry has previously been shown to be the best predictor of functional antibodies for CAR generation, this method was used to identify potential candidate antibodies from over 7 fusions performed in the laboratory. A typical flow cytometry screen of hybridomas positive by initial ELISA screen using LHR-Fc coated plates is shown below in FIG. 4 using the ES-2 ovarian carcinoma cell line. As seen in this figure with hybridoma 8B7, only rare LHR hybridomas were shown to produce high MFI by flow cytometry. These few candi...
example 2
Monoclonal Antibodies Detecting the Expression of LHR in Ovarian Cancer
[0280]The overall hypothesis is that ovarian cancer can be treated effectively and safely with LHR chimeric antigen receptor modified T-cells. As a target, LHR has significant advantages over other targets due to its common expression on ovarian cancers and its lack of expression on normal human tissues. LHR CAR T-cells are produced in vitro and in vivo to identify a potential clinical candidate for subsequent clinical trials or use with dual targeting CAR modified T-cells.
Construction and Synthesis Single Chain LHR Antibody Genes
[0281]The DNA sequences for the 5 high binding anti-LHR antibodies (8B7-3, 5F4-21, 5B1-1, 2H11-37, and 138-2) were sequenced by MCLAB (South San Francisco, Calif.). All five antibodies are tested to determine which one produces the most effective CAR in assays described below. As shown below in FIG. 6, third generation CAR vectors were constructed consisting of the following tandem genes...
example 3
CAR T-Cells
Construction of the CAR Lentiviral Constructs
[0291]The CAR consists of an extracellular antigen binding moiety or scFV which binds LHR. The scFV is connected via a CD8 hinge region to the cytoplasmic signaling domain, comprised of the CD8 transmembrane region, and the signaling domains from CD28, 4-1BB and CD3z. The entire CAR sequence including the signaling domains, were synthetically synthesized by Genewiz Gene Synthesis Services (Piscataway, N.J.) (FIG. 10). The plasmids are purified and digested with the appropriate restriction enzymes to be inserted into an HIV-1-based, bicistronic lentiviral vector (pLVX-IRES-ZsGreen, Clontech, Signal Hill, Calif.) containing HIV-1 5′ and 3′ long terminal repeats (LTRs), packaging signal (Ψ), EF1α promoter, internal ribosome entry site (IRES), woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and simian virus 40 origin (SV40) via overnight T4 DNA ligase reaction (New England Biosciences; Ipswich, Mass.). Nov...
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