Method for preparing antibodies having improved properties
An antibody and performance technology, applied in the field of production of Fc-containing polypeptides, which can solve problems such as reduced affinity
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Embodiment 1
[0170] Construction of Anti-TNFαFc Mutant Protein
[0171] Fc containing 2 mutations (F243A / V264A) in the anti-TNF monoclonal antibody was made in Pichia pastoris using the sequence and protocol listed below. The heavy and light chain sequences of the parental (wild type) anti-TNFα antibody are set forth in SEQ ID NO: 1 and 2. The sequence of the heavy chain of the double mutein anti-TNFα antibody is set forth in SEQ ID NO:3. The light chain sequences of wild-type and double mutein anti-TNFα antibodies were identical.
[0172] The signal sequence of the alpha-mating factor prodomain (SEQ ID NO: 4 and 5) was fused in frame to the end of the light or heavy chain by PCR fusion. The sequence was codon-optimized and synthesized by Genscript (GenScript USA Inc., 860 Centennial Ave. Piscataway, NJ 08854, USA). In a similar manner to the construction of anti-HER2 IgGl and its Fc mutein, both the heavy and light chains were cloned into antibody expression vectors.
[0173] The heav...
Embodiment 2
[0212] Antitumor activity of α-2,3 sialylated FC-containing polypeptides
[0213] To determine whether α-2,3-linked sialylation of Fc-containing polypeptides could enhance effector functions of immune cells, the effect of α-2,3-linked SA IgG was determined using the 4T1 tumor cell line.
[0214] The mouse mammary tumor cell line 4T1 [ATCC CRL-2539] stably transfected with firefly luciferase [Luc2] was cultured in RPMI-1640 medium supplemented with 10% FBS. 3 x 10 5 4T1-Luc2 cells. One week after implantation, tumors were evaluated by three-dimensional measurements using a Biopticon TumorImager and randomized into treatment groups. Groups of 5 mice each were treated with the indicated doses of antibodies in a weekly treatment regimen for 3 consecutive weeks. Tumor volumes were monitored weekly and the results analyzed using GraphPad Prism software.
[0215] In this model, an anti-mouse PD1 antagonistic antibody (prepared in-house) was used as a positive control. Isotype an...
Embodiment 3
[0220] Adjuvant and antitumor activity of anti-CD40 agonistic antibodies with increased amounts of α2,3 sialic acid
[0221]CD40 is a member of the tumor necrosis factor receptor (TNFR) superfamily expressed on antigen presenting cells. CD40 agonists have been shown to trigger immune responses against different tumors and inhibit the growth of different neoplastic cells in vitro and in vivo. It has been demonstrated that agonistic mAbs against CD40, which have enhanced binding to the Fcγ receptor IIB on antigen-presenting cells, increase the activation of antigen-presenting cells and thereby promote adaptive immune responses (Li and Ravetch, science 333(6045):1030 (2011)). It has been proposed that agonistic CD40 antibodies require coengagement of inhibitory FcgRIIB, resulting in the maturation of DCs that promote the proliferation and activation of cytotoxic CD8+ T cells.
[0222] To investigate whether an agonistic anti-CD40 mAb with increased Fcγ receptor IIB binding co...
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