Bi- or multispecific polypeptides that bind immune effector cell surface antigens and hbv antigens for the treatment of hbv infection and related disorders
A surface antigen, antigen technology, applied in the field of peptides
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Embodiment 1
[0120] Materials and methods used in Example 2
[0121] Cloning and preparation of bispecific antibodies
[0122] Variable heavy genes encoding anti-CD3 (OKT3), anti-CD28 (9.3), anti-CD16 (A9) and anti-CD56 (NCAM29.2) were obtained by PCR amplification from reverse transcribed mRNA from individual hybridomas. chain and variable light chain complementary DNA using a set of primers covering all VH and Vk / Vl subtypes. The PCR product was ligated into pCR2.1-TOPO (Invitrogen, Life Technologies) and sequenced. Anti-HBsAg scFvC8 is provided in codon-optimized form in plasmid pMP71-C8. The variable heavy and variable light chain cDNAs encoding the above antibodies were combined into scFvs with glycine-serine linkers using primers containing appropriate restriction sites on the 5' and 3' sides. OKT3, 9.3, A9 and NCAM29.2 scFvs (N-terminal extended (Gly) 3-4 ) was cloned into the 3' end of the cDNA encoding the Fc domain (hinge, CH2, CH3) of human IgG1 presented in pBluescript KS...
Embodiment 2
[0138] result
[0139] In the first-line experiments, we assessed the activity of bispecific antibody constructs against the surface antigens CD3 and CD28 on CTLs and against the surface antigens CD16 and CD56 on NK cells. We utilized plasmid-transfected HBV surface antigen-producing hepatoma cell lines. After HBV protein expression was established, these target cells were co-cultured with immune effector cells (ie, PBMCs and isolated NK cells) and bispecific antibody constructs. PBMCs contain approximately 70% T cells but only 7% NK cells. Therefore, we magnetically isolated CD16 + CD56 + NK cells. As a negative control we analyzed co-cultures with HBV-negative target cells that had been pre-incubated with supernatant containing HBV and subviral particles. We used this control to rule out activation of effector cells due to nonspecific binding to HBV particles on the surface of HBV-negative target cells. Additionally, we co-cultured HBV-positive target cells with immu...
Embodiment 3
[0146] Method for Example 4
[0147] To analyze the therapeutic potential of bispecific antibody constructs to successfully retarget T cells to HBV-positive cells, in vitro co-culture experiments were performed and analyzed in detail. We utilized bispecific antibody constructs containing single-chain binding domains against human CD3 (αCD3) and human CD28 (αCD28), and additionally in their F c Constructs containing directed mutations in the spacer domain (ΔADCC) that abolish antibody-dependent cellular cytotoxicity (by avoiding Fcγ receptor binding). These are constructed as a safety measure to rule out non-specific activation of natural killer cells. On the other side, all bispecific antibody constructs carried the HBV S-protein (HBsAg) specific binding domain C8. Peripheral blood mononuclear cells (PBMC) isolated from fresh venous blood of healthy donors were co-cultured with different human hepatoma cell lines for surrogate HBV-infection. We used HuH7-S (HBV S-antigen ...
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