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Antibody targeting AXL and antibody-drug conjugate and preparation method and application thereof

An antibody drug and conjugate technology, applied in the direction of antibodies, drug combinations, anti-tumor drugs, etc., can solve the problems of changes in the safety and efficacy of ADC drugs, and the safety and efficacy cannot be predicted before experiments.

Inactive Publication Date: 2019-11-22
FUDAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] 3) The characteristics of the linker: the linker needs to be stable enough in the blood, and at the same time, the change of the linker will change the number of linked drugs and the location of the area on the ADC, which will eventually lead to changes in the safety and effectiveness of the entire ADC drug
[0011] It can be seen that the development of ADC drugs requires a lot of experimental exploration and verification, and its safety and effectiveness cannot be predicted before the experiment.

Method used

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  • Antibody targeting AXL and antibody-drug conjugate and preparation method and application thereof
  • Antibody targeting AXL and antibody-drug conjugate and preparation method and application thereof
  • Antibody targeting AXL and antibody-drug conjugate and preparation method and application thereof

Examples

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preparation example Construction

[0239] Antibody preparation

[0240] The sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by conventional techniques, such as PCR amplification or genomic library screening. In addition, the coding sequences for the light and heavy chains can be fused together to form single-chain antibodies.

[0241] Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

[0242] In addition, related sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.

[0243] At present, the DNA sequence encoding the antibody of the ...

Embodiment 1

[0328] Example 1 Discovery and Preparation of Monoclonal Antibody Targeting Human AXL

[0329] Step 1. Preparation of hybridoma cells:

[0330] First, the extracellular region of human AXL protein (AXL-ECD) was prepared as an antigen. Referring to the 33rd to 449th amino acids of NCBI: NP_068713.2, a C-terminus polyhistidine-tagged antigen was obtained using gene cloning technology and a mammalian vector expression system. The specific amino acid sequence is as follows ( SEQ ID NO.: 36):

[0331] QAEESPFVGNPGNITGARGLTGTLRCQLQVQGEPPEVHWLRDGQILELADSTQTQVPLGEDEQDDWIVVSQLRITSLQLSDTGQYQCLVFLGHQTFVSQPGYVGLEGLPYFLEEPEDRTVAANTPFNLSCQAQGPPEPVDLLWLQDAVPLATAPGHGPQRSLHVPGLNKTSSFSCEAHNAKGVTTSRTATITVLPQQPRNLHLVSRQPTELEVAWTPGLSGIYPLTHCTLQAVLSDDGMGIQAGEPDPPEEPLTSQASVPPHQLRLGSLHPHTPYHIRVACTSSQGPSSWTHWLPVETPEGVPLGPPENISATRNGSQAFVHWQEPRAPLQGTLLGYRLAYQGQDTPEVLMDIGLRQEVTLELQGDGSVSNLTVCVAAYTAAGDGPWSLPVPLEAWRPGQAQPVHQLVKEPSTPAFSWP HHHHHHHHHH

[0332] The AXL ectodomain protein prepared above was...

Embodiment 2

[0341] Example 2 Antibody Sequencing, Identification of Complementarity Determining Regions (CDRs)

[0342] Based on excellent specificity and affinity, mAb001, mAb002, mAb005, and mAb006 were preferentially selected for antibody sequencing identification. Primers were designed to amplify heavy chain (VH) and light chain (VL) variable region fragments by conventional PCR techniques (see figure 2 ), cloned into the vector, and sequenced. Using conventional sequencing and analyzing the Kabat database (http: / / www.bioinf.org.uk), the following heavy chain variable region (VH), light chain variable region (VL) amino acid sequence, complementarity determining region (CDR) information was obtained , the underline "" shows the amino acid sequence of CDR-1 / 2 / 3. After gene sequencing, it was noted that the CDR sequences of mAb006c and mAb005c are highly similar and are no longer listed separately.

[0343] SEQ ID NO.: 7mAb002 heavy chain variable region (VH) amino acid sequence

[...

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Abstract

The invention discloses a novel monoclonal antibody targeting AXL and an antibody-drug conjugate. The invention further discloses a preparation method of the antibody and the antibody-drug conjugate.The AXL antibody can efficiently and highly specifically bind purified human AXL proteins and AXL on the surfaces of various tumor cells. The humanized antibody further has high affinity and low immunogenicity. The AXL antibody-drug conjugate has significant anti-tumor effects on tumor cells with high expression of AXL.

Description

technical field [0001] The present invention relates to the field of medicines, more specifically, to AXL-targeted antibodies, antibody-drug conjugates, preparation methods and uses thereof. Background technique [0002] AXL is a member of receptor tyrosine kinase subfamily-TAM family, TAM family includes Tyro-3, Axl and Mer, and their ligands are protein molecules encoded by growth arrest specific gene 6 (Gas6). AXL is activated after binding to Gas6, thereby activating its downstream signal transduction pathways such as PI3K / AKT, RAS / ERK and β-Catenin / TCF, thereby regulating cell proliferation, apoptosis, chemotaxis, adhesion and recognition, etc. Physiological function. [0003] Studies have found that AXL is activated in a variety of cancers, such as lung cancer, breast cancer, prostate cancer, thyroid cancer, endometrial cancer, ovarian cancer and kidney cancer, and participates in the epithelial-mesenchymal transition of tumor cells, Various mechanisms such as angiog...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K5/10G01N33/68A61K47/68A61P35/00
CPCC07K16/2863G01N33/6854A61K47/6801A61P35/00C07K2317/565C07K2317/622C12N5/0634G01N33/573G01N2333/91205A61K47/6877A61K47/6849A61K47/6889C07K2317/24C07K2317/92C07K2317/77C07K2317/33A61K47/68031A61K45/06A61K2039/505A61K47/6817
Inventor 余科沈竞康孟韬马兰萍王昕金锐裴金鹏杜志彦陈驎于霆张永良
Owner FUDAN UNIV
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