Anti-CAIX (MSC8) h(CD28-CD3ζ) CAR, pMMLV vector

All products and services are For Research Use Only and CANNOT be used in the treatment or diagnosis of disease.

Recombinant Moloney murine leukemia virus (MMLV) retroviral vectors serve as efficient viral vector tools for introducing genes permanently into a wide variety of dividing cells, and provide an alternative to modify primary T cells. Creative biolabs has developed retroviral CAR vector pMMLV CAIX (MSC8) h(28ζ), which is constructed for the engineering of T cells to target human CAIX. The T cells are genetically modified through transduction with a retroviral vector expressing scFv of anti-CAIX antibody linked to a CD28 transmembrane domain and CD3-zeta signaling domains. And the vector product was designed for the treatment of Renal cell carcinoma.

Details

Target
CAIX

Targeting Cell Type
T cell

Targeting Diseases
Renal cell carcinoma

Generation
Second

Vector Name
pMMLV

Vector Length
~6kb

Vector Type
Recombinant Moloney murine leukemia virus (MMLV) retroviral vector

Receptor Construction
scFv-CD28-CD3ζ

Discription of Signaling Cassetes
CD28
CD28 (Cluster of Differentiation 28) is one of the proteins expressed on T cells that provide costimulatory signals required for T cell activation and survival. CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2) proteins which are expressed on antigen-presenting cells (APC). CD28 modulates the primary TCR/CD3ζ signal in a different fashion than the late costimulatory elements OX40 and 4-1BB. CD28 enhances the expression of downstream regulators that impact on T-cell proliferation, death, differentiation, and effector functions. CAR+ T cells containing the CD28 endodomain showed strikingly enhanced sustained T cell activation, growth, survival. And CD28 results in a brightly expressed, stable receptor as the transmembrane domain. Including CD28 costimulatory domains in CARs led to enhanced anti-malignancy efficacy.
CD3ζ
CD3ζ, also known as T-cell receptor zeta, which together with T-cell receptor and CD3γ, δ, ε chain, forms the TCR-CD3 complex. ζ was expressed independently from the complex. The zeta chain plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways. CD3-zeta, which contains 3 ITAMs, is the most commonly used endodomain component of CARs. It transmits an activation signal to the T cell after antigen is bound. CD3-zeta may not provide a fully competent activation signal and additional co-stimulatory signaling is needed. For example, chimeric 4-1BB and OX40 can be used with CD3-zeta to transmit a proliferative/survival signal, or all three can be used together.

Target

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Published Data

Surface plasmon resonance

Fig.1 Serial dilutions of purified Fab were injected on a rhCAIX-coated CM5 sensor chip. Values were corrected for binding to the reference flow cell. The solid lines represent the theoretical curves for each Fab concentration calculated according to the 1 : 1 binding model. KA, ¾ and KD values calculated by the 1 : 1 binding model are shown in the table.

CAR Construction :

FCM

Fig.2 Selection of CAIX-specific antibodies.

CAR Construction :

Fig.2 Selection of CAIX-specific antibodies.

Antibody binding to (A) wild-type (dashed) or CAIX-transfected (shaded) SKRC 17 cells, (B) HeLa cells under normoxic (dashed) or hypoxic (shaded) conditions and (C) murine CT26 cells under normoxic (dashed) or hypoxic (shaded) conditions was studied by flow cytometry. Sample analyses are shown for SC3 and MSC8 Fab antibody fragments. Fab ESC 1 1 recognizing human fibroblast activation protein and antibodies against either human (M75) or murine CAIX (M- 100) served as controls.

ELISA

Fig.3 Antibody binding to carbonic anhydrase isoforms II, IV, IX, XII and XIV was analyzed in ELISA on recombinant proteins.

CAR Construction :

Fig.3 Antibody binding to carbonic anhydrase isoforms II, IV, IX, XII and XIV was analyzed in ELISA on recombinant proteins.

ESC1 1 IgG recognizing human fibroblast activation protein served as negative control. Coating was confirmed by staining with a Penta-His antibody.

FunsC

Fig.2 In vitro carbonic anhydrase activity assay on HCTl 16 membrane fragments.

CAR Construction :

Fig.2 In vitro carbonic anhydrase activity assay on HCTl 16 membrane fragments.

(A) Time course for C02 hydration with membrane-free buffer (dotted), HCT l 16-EV membrane-fragments (grey), HCTl 16-CAIX membrane fragments (black) and HCTl 16-CAIX membrane fragments blocked with MSC 8 Fab (dashed). (B) Carbonic anhydrase activity, normalized to a scale where activity measured in HCTl 16-CAIX and HCTl 16-EV membrane fragments are one and zero, respectively. Asterisks denote significant reduction in activity (MSC5, MSC 10„MSC12 and MSC8 Fab antibodies). (C) Time course for CO2 hydration with HCTl 16-EV membrane-fragments (grey), HCTl 16-CAIX membrane- fragments (black) and HCTl 16-CAIX membrane fragments blocked with MSC3 IgG (dashed black), MSC8 IgG (dotted black) or acetazolamide (dashed grey). (D) Dose-response curves for MSC 8 Fab (grey) and MSC 8 IgG (black) influence on CAIX-activity (using the same normalization algorithm as in B).

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For research use only. Not intended for any clinical use. No products from Creative Biolabs may be resold, modified for resale or used to manufacture commercial products without prior written approval from Creative Biolabs.