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Antigen binding molecules comprising a TNF family ligand trimer and pd1 binding moiety

An antigen-binding molecule, trimer technology, applied in the direction of NGF/TNF-superfamily, receptor/cell surface antigen/cell surface determinant, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc. , which can address issues such as known elevated toxicity

Active Publication Date: 2019-04-02
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Administration of a combination of an anti-PD1 antibody and a 4-1BB agonist antibody in a tumor mouse model resulted in a potent long-lasting systemic antitumor response, whereas the known toxicity of the 4-1BB agonist alone was slightly elevated (Chen et al. ,2014)

Method used

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  • Antigen binding molecules comprising a TNF family ligand trimer and pd1 binding moiety
  • Antigen binding molecules comprising a TNF family ligand trimer and pd1 binding moiety
  • Antigen binding molecules comprising a TNF family ligand trimer and pd1 binding moiety

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0719] 1.1 Generation of anti-PD-1 antibodies

[0720] Immunization of mice

[0721] Genetic immunization was performed on NMRI mice by using a plasmid expression vector encoding full-length human PD1 by intradermal application of 100ug vector DNA (plasmid 15300_hPD1-fl), followed by electroporation (2 1000V / cm rectangular pulses, lasting 0.1ms, 0.125s interval; then 4 rectangular pulses of 287.5V / cm, duration 10ms, interval 0.125s). Mice received 6 consecutive immunizations on 0, 14, 28, 42, 56, 70, and 84 days. Blood was collected and serum was prepared on days 36, 78 and 92 for titer determination by ELISA (see below). The animal with the highest titer was selected for enhancement by intravenous injection of 50ug of recombinant human PD1 human Fc chimera on day 96, and monoclonal antibodies were isolated by hybridoma technology by fusing splenocytes with myeloma cell lines 3 days after enhancement .

[0722] Determination of serum titer (ELISA)

[0723] Immobilize human recombi...

Embodiment 2

[0809] Preparation of PD1 targeting Fc fusion antigen binding molecule containing 4-1BB ligand trimer

[0810] According to the P41273 sequence (SEQ ID NO: 103) of the Uniprot database, different fragments of the DNA sequence encoding the partial outer domain (amino acids 71-254 and 71-248) of the human 4-1BB ligand were synthesized.

[0811] 2.1 Monovalent PD1 (0314) with crossed CH1-CL domain with charged residues targeting Fc (kih) fusion antigen binding molecule containing 4-1BB ligand (71-254) trimer (construction 7.1) Preparation

[0812] The clone contains pass (G 4 S) 2 The two outer domains (71-254) of the 4-1BB ligand separated by the linker and the polypeptide fused to the human IgG1-CL domain, such as Image 6 Depicted in A: human 4-1BB ligand, (G 4 S) 2 Connector, human 4-1BB ligand, (G 4 S) 2 Connect the head, people CL.

[0813] Clone a polypeptide that contains an outer domain (71-254) of 4-1BB ligand and is fused with human IgG1-CH1 domain, such as Image 6 Described ...

Embodiment 3

[0934] Production and purification of monovalent and bivalent anti-PD1 targeting split-trimer 4-1BB ligand Fc fusion antigen binding molecules and control molecules

[0935] The targeted and non-targeted split trimer 4-1BB ligand Fc (kih) fusion molecule coding sequence is cloned into a plasmid vector, which drives the expression of the insert from the MPSV promoter and contains the synthesis at the 3'end of the CDS polyA sequence. In addition, the vector contains the EBV OriP sequence used for plasmid episomal maintenance.

[0936] The split trimer 4-1BB ligand Fc (kih) fusion molecule was generated by co-transfecting HEK293-EBNA cells with a mammalian expression vector using polyethyleneimine. Transfect the cells with the corresponding expression vector. For constructs 7.1, 7.2, 7.4, 7.6 and the corresponding control molecule controls B and D, at a ratio of 1:1:1:1 ("Carrier Fc Hole Chain": "Carrier PD1 Light Chain": "Carrier Fc Segment Chain" : "Carrier 4-1BBL light chain"). ...

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Abstract

The invention relates to novel TNF family ligand trimer-containing antigen binding molecules comprising (a) at least one moiety capable of specific binding to PD1 and (b) a first and a second polypeptide that are linked to each other by a disulfide bond, characterized in that the first polypeptide comprises two ectodomains of a TNF ligand family member or fragments thereof that are connected to each other by a peptide linker and in that the second polypeptide comprises only one ectodomain of said TNF ligand family member or a fragment thereof.

Description

Invention field [0001] The present invention relates to novel antigen-binding molecules containing TNF family ligand trimers, which comprise (a) at least one module capable of specifically binding PD1 and (b) first and second polypeptides connected to each other by disulfide bonds , Wherein the antigen-binding molecule is characterized in that the first polypeptide comprises two outer domains or two fragments of a TNF ligand family member connected to each other by a peptide linker and in that the second polypeptide comprises a TNF ligand family member Only one foreign domain or fragments thereof. The invention further relates to methods of generating these molecules and methods of using them. Background of the invention [0002] Ligands that interact with molecules of the TNF (tumor necrosis factor) receptor superfamily play a key role in the organization and function of the immune system. While regulating normal functions such as immune response, hematopoiesis and morphogenes...

Claims

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Application Information

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IPC IPC(8): C07K14/705A61K39/395C07K16/28C12N15/62
CPCC07K14/70575C12N15/62C07K2317/24C07K2317/71C07K2317/76C07K2317/92C07K2319/00C07K2319/74C07K2319/75C07K16/2818A61P35/00C07K2317/55
Inventor C·费拉拉科勒C·克劳斯C.克雷恩S·西伯M·阿曼S·格劳-理查兹P·布鲁恩克P·乌玛纳V·列维茨基E·莫斯纳
Owner F HOFFMANN LA ROCHE & CO AG
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