Novel ICOS antibodies and tumor targeting antigen binding molecules comprising them
A technology of antigen-binding molecules and tumor-associated antigens, which can be applied to anti-tumor drugs, anti-receptors/cell surface antigens/cell surface determinant immunoglobulins, antibody medical components, etc., and can solve the problem of insufficient closing brakes
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[0464] The following are examples of methods and compositions of the present invention. It should be understood that various other embodiments may be practiced given the general description provided above.
[0465] recombinant DNA technology
[0466] Standard methods are used to manipulate DNA, as described in Sambrook et al., Molecular cloning: Alaboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biology reagents were used according to the manufacturer's instructions. General information on the nucleotide sequences of human immunoglobulin light and heavy chains is given in the following reference: Kabat, E.A. et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication No 91- 3242.
[0467] DNA sequencing
[0468] DNA sequence was determined by double-stranded sequencing.
[0469] gene synthesis
[0470] The desired gene segments were generated by PCR using appropriate templates or synth...
example 1
[0485] Generation of ICOS antibodies
[0486] 1.1 Preparation, purification and characterization of antigens and screening tools for the generation of novel ICOS conjugates by immunization
[0487] 1.1.1 Preparation, purification and characterization of monomeric and dimeric ICOS antigen Fc(kih) fusion molecules
[0488] DNA sequences encoding the ectodomains of human, cynomolgus or mouse or 4-1BB (Table 1) were subcloned in frame with the human IgG1 heavy chain CH2 and CH3 domains in the knob of the monomer as well as the dimeric ICOS antigen Fc Hole and pestle of fusion molecules (Merchant et al., 1998). An Avi tag was introduced at the C-terminus of the antigenic Fc protrusion for targeted biotinylation. Combination of an antigenic Fc knob chain containing S354C / T366W mutations with an Fc hole chain containing Y349C / T366S / L368A / Y407V mutations allows the production of heterodimers containing a single copy of ICOS or homodimers containing two copies of an ectodomain-contai...
example 2
[0561] Characterization of anti-ICOS antibodies
[0562] 2.1 Binding on human ICOS
[0563] 2.1.1 Surface Plasmon Resonance (Affinity + Affinity)
[0564] Binding of immunogenic ICOS-specific antibodies to recombinant monomeric ICOS Fc(kih) was assessed by surface plasmon resonance (SPR). All SPR experiments were performed on a Biacore T200 at 25°C using HBS-EP as running buffer (0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.005% Surfactant P20, Biacore, Freiburg / Germany).
[0565] Kinetic constants were derived using Biacore T200 evaluation software (vAA, Biacore AB, Uppsala / Sweden) to fit rate equations for 1:1 Langmuir binding by numerical integration and used to qualitatively estimate affinities.
[0566] In the same experiment, the affinity of the interaction between immunogenic ICOS-specific antibody molecules 8, 14, 18 and 20 and recombinant human ICOS was determined. For this purpose, the ectodomain of human ICOS was subcloned using the avi (GLNDIFEAQKIEWHE) tag (see ...
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