Antibodies that specifically bind pd-1 and methods of use
A PD-1, PD-L1 technology, applied in the direction of antibodies, antibody medical components, chemical instruments and methods, etc., can solve problems such as adequate response to treatment
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[0702] Preparation of immunogenic antigens and production of monoclonal antibodies can be performed by any suitable technique such as recombinant protein production. Immunogenic antigens can be administered to animals in the form of purified proteins or protein mixtures, including whole cells or cell extracts or tissue extracts, or antigens can be formed de novo in animals from nucleic acids encoding the antigens or portions thereof.
[0703] In some embodiments, an antibody or antigen-binding fragment thereof of the invention that specifically binds PD-1 is a bispecific antibody.
[0704] In some embodiments, an antibody or antigen-binding fragment thereof of the invention is a multispecific antibody.
[0705] The monospecific antibodies provided herein that specifically bind to PD-1 can be engineered into bispecific antibodies, which are also encompassed within the scope of the present invention.
[0706] Full-length bispecific antibodies can be produced, for example, using...
Embodiment 1
[0906] Embodiment 1. Method
[0907] Affinity measurements using surface plasmon resonance (SPR)
[0908] Affinity measurements were performed using the ProteOn XPR36 system (BioRad). Anti-human IgG Fc (Jackson cat. no. 109-005-098) was coupled to the surface of the modified alginate polymer layer of a GLC chip (BioRad, cat. no. 176-5011 ) by using the manufacturer's instructions for the amine coupling chemistry to prepare the biosensor surface. Approximately 5000 RU (response units) of mAb were immobilized. Kinetic experiments were performed in running buffer (DPBS + 0.01% P20 + 100 μg / mL BSA) at 25°C. For kinetic experiments, 200 RU of mAb were captured and then injected with analytes (human and cynomolgus monkey PD-1 ) at concentrations ranging from 1.563 nM to 400 nM (in 4-fold serial dilutions). The association phase was monitored at 50 μL / min for 3 minutes, followed by 10 or 15 minutes of buffer flow (dissociation phase). At 100 μL / min, with 100 mM H 3 PO 4 (S...
Embodiment 2
[0930] Example 2. Production and structural characterization of agonistic antibodies specifically binding to PD-1
[0931] Three Balb / c mice and three Balb / c mice were immunized with the extracellular domain (ECD) of human PD-1 (SEQ ID NO: 1) (huPD-1-Fc) conjugated to Fc and hybridomas generated using standard protocols. C3H mice. Hybridomas were screened for binding to recombinant PD-1 (ECD) by ELISA. A hit was defined as a sample with an ELISA signal five times the mean of the negative control. Positive clones were cross-screened against an irrelevant Fc fusion protein and bound mouse PD-1. Supernatants from single-cell cloned hybridomas were tested for binding to human PD-1 and cynomolgus PD-1 proteins. A hit was defined as a signal greater than the mean +3 S.D. of the negative control.
[0932] Selected mouse antibodies were cloned as chimeric mAbs into human IgG1 and tested for their ability to inhibit antigen-specific T cell activation in a CMV recall assay (CMV-PB...
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