Immunostimulatory compositions and methods
A conjugate and co-stimulation technology, applied in pharmaceutical formulations, animal/human proteins, receptors/cell surface antigens/cell surface determinants, etc., can solve unsatisfactory clinical responses, adverse side effects, and lack of tumor specificity And other issues
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Embodiment 1
[0228] Example 1: CSA-4-1 BBL amplifies the response generated by the same antigen
[0229] As mentioned above, 4-1 BBL plays an important role in the regulation of acquired and innate immune responses. 4-1 BBL is used as a costimulatory molecule to activate CD4 + And CD8 +T cells, NK cells and DC, and inhibit T reg Suppressive function of cells. Therefore, the molecule can be used as a specific adjuvant to generate an effective tumor response for cancer treatment.
[0230] Due to the presence of the core streptavidin part, the CSA-4-1 BBL fusion protein forms a tetrameric / oligomeric structure and is a soluble molecule. The immunostimulatory activity of CSA-4-1 BBL on T cell responses was demonstrated using the alloantigen mixed lymphocyte reaction (MLR), as described below.
[0231] In the presence or absence of CSA-4-1 BBL, C57BL / 6 mouse lymph node cells were used as a responder against BALB / c irradiated splenocytes. In the last 18 hours of the incubation period, use [ 3 H] The ...
Embodiment 2
[0232] Example 2 : CSA-4-1 BBL improves T cell proliferation
[0233] In order to determine the relative activity of the 4-1-BBL fusion protein and the monoclonal antibody against 4-1BB, in the proliferation test, in the presence of various contents of the 4-1 BBL fusion protein and antibody, a suboptimal concentration of The anti-CD3 antibody will be CD4 sorted by flow cytometry + And CD8 + Polyclonal stimulation of T cells. The activity of the fusion protein on T cell proliferation is 70-fold higher than that of the antibody ( Picture 9 ).
[0234] Because the antibody Ab has been shown to have effective activity in animal models of cancer immunotherapy. See, for example, Melero et al., 1998, Cell Immunol. 190: 167-72; Melero et al., 1997 Nat. Med. 3: 682-85. The data indicate that CSA-4-1BBL fusion protein will be a useful component of cancer vaccines. Adjuvant or as a carrier to deliver TAA to DC.
Embodiment 3
[0235] Example 3 : Biotinylated OVA / CSA-4-1BBL conjugate vs. CD8 + The role of T cells
[0236] Ovalbumin peptide (OVA) was biotinylated using a commercially available kit (Pierce Biotechnology, Rockford, IL). The biotinylated OVA was pre-mixed with CSA-4-1BBL fusion protein in different ratios in vitro for conjugation, and one million OT-1T cells were injected intraperitoneally into the acquired transfer of natural C58BL / 6. SJL animals. Specifically, one million OT-I CD8s are marked with CFSE + T cells were transferred to B6.SJL mice, which were treated with biotinylated ovalbumin (10μg / injection) ("OVA") and CSA-4-1BBL (1μg / injection) mixed with biotinylated OVA. Injection) ("41BBL+OVA") or conjugated OVA-Biotin / CSA-4-1 BBL ("41BBL-OVA") immunization. ( Picture 10 ) Picture 10 The last picture ("41 BBL-OVA") shows the response to 5 μg CSA-4-1 BBL conjugated with 10 μg biotinylated OVA. For comparison, core streptavidin ("SA") was used at an equimolar level with CSA-4-1BBL.
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