Method and drug combination for treating disease with anti-tigit antibody combined with PD-1 inhibitor
A technology of PD-1 and inhibitors, applied in drug combinations, antibody medical components, antibodies, etc., can solve problems such as disappearance
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Embodiment 1
[0174] Example 1. Preparation of TIGIT antigen antibody
[0175] 1.1 Protein design and expression
[0176] Using human TIGIT protein (Uniprot No.: Q495A1) as the template of TIGIT of the present disclosure, the amino acid sequences of the antigen and detection protein of the present disclosure were designed, and different tags were optionally fused on the basis of the TIGIT protein, and cloned into the pHr carrier ( Self-produced) or pXC-17.4 vector (LONZA), transiently expressed in 293 cells or stably expressed in CHO cells and purified to obtain the antigens and detection proteins encoding the disclosure. The following TIGIT antigens without special instructions refer to human TIGIT.
[0177] Fusion protein of TIGIT extracellular domain and mouse IgG2aFc fragment: TIGIT-mFc for immunization and detection
[0178] MEFGLSWLFFLVAILKGVQC MMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPEPRGPTIKPCPPCKCPAP...
Embodiment 2
[0197] Example 2. Preparation of anti-human TIGIT hybridoma monoclonal antibody
[0198] 2.1 Immunization
[0199] Anti-human TIGIT monoclonal antibodies were produced by immunizing mice. SJL white mice were used in the experiment, female, 6-8 weeks old (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., animal production license number: SCXK (Beijing) 2012-0001). Breeding environment: SPF grade. After the mice were purchased, they were reared in a laboratory environment for 1 week, regulated by a 12 / 12 hour light / dark cycle, the temperature was 20-25°C, and the humidity was 40-60%. The acclimated mice were immunized according to the following protocol. The immunizing antigen was the extracellular region of human TIGIT with mFc (SEQ ID NO: 1).
[0200] Immunization regimen: with Gold Adjuvant (Sigma Cat No.T2684) with Thermo Alum (Thermo Cat No. 77161) adjuvant cross-immunization. Antigen and adjuvant ( Gold Adjuvant) ratio of 1:1, antigen and adjuvant (T...
Embodiment 3
[0242] Example 3. Humanization of murine anti-human TIGIT antibody
[0243] By comparing the IMGT human antibody heavy and light chain variable region germline gene database with MOE software, the heavy chain and light chain variable region germline genes with high homology to the mouse antibody were selected as templates, and the murine antibody's germline genes were selected as templates. The CDRs were grafted into corresponding human templates to form variable region sequences in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. If necessary, back-mutate the amino acids in the FR region to obtain a humanized anti-TIGIT monoclonal antibody. In the following exemplary embodiments, the determination of amino acid residues in the CDR regions is determined and annotated by the Kabat numbering system.
[0244] The light and heavy chain variable regions of the above murine antibodies are linked with the light and heavy chain constant regions of human antibodies to form a chimeric anti...
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