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Humanized universal light chain mice

A humanized, mouse technology, applied in the field of humanized universal light chain mice, can solve problems such as unfavorable expression or binding performance of two heavy chains, and no solution.

Active Publication Date: 2014-07-09
REGENERON PHARM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But there is no easy solution for selecting light chains that can efficiently associate and express heavy chain heterodimers
It is certainly possible to develop human light chain variable domains in humanized mice for therapeutic use in humans, but selecting a light chain that efficiently binds and expresses a heavy chain with the desired binding properties is not straightforward. Solutions where the light chain does not favor the expression or binding properties of the two heavy chains

Method used

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Examples

Experimental program
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Effect test

Embodiment I

[0239] Humanization of mouse immunoglobulin genes

[0240] Thirteen different BAC targeting vectors (BACvec) were engineered using human and mouse bacterial artificial chromosomes (BACs) to humanize mouse immunoglobulin heavy chain and kappa light chain loci. Tables 1 and 2 give a detailed description of the steps performed to construct all BACvecs for humanization of the mouse immunoglobulin heavy chain and kappa light chain loci, respectively.

[0241] Identification of human and mouse BACs

[0242] Mouse BACs spanning the 5' and 3' ends of the immunoglobulin heavy chain and kappa light chain loci were identified by hybridization with filters of the BAC library or by PCR screening of the mouse BAC library DNA pool. Using a probe corresponding to the region of interest, hybridize the filter under standard conditions. The library pool is screened by PCR using unique primer pairs flanking the targeted region of interest. Additional PCRs were performed using the same primers ...

Embodiment II

[0272] Generation of fully humanized mice by combining multiple humanized immunoglobulin alleles

[0273] ES cells carrying a portion of the human immunoglobulin heavy chain or kappa light chain variable repertoire as described in Example 1 were microinjected at several locations and the resulting mice were bred to produce various types of Humanized mice with progressively increasing proportions of the human germline immunoglobulin repertoire (Table 5; Figure 5A and 5B ). 1 (V1) humanized mice have 18 human V H Gene segments and all human D H and J H gene segments, as well as 16 human Vκ gene segments and all human Jκ gene segments. 2 (V2) humanized mice and (V3) humanized mice have an increased variable lineage with a total of 39 V H and 30 Vκ, and 80 V H and 40 Vκ. Because encoding mouse V H 、D H and J H gene segments and the genomic regions of the Vκ and Jκ gene segments have been completely replaced, so any version of Antibodies produced by humanized mic...

Embodiment III

[0278] Lymphocyte populations in mice with humanized immunoglobulin genes

[0279] Three different formats were evaluated by flow cytometry Mature B cell populations in mice.

[0280] Briefly, cell suspensions from bone marrow, spleen and thymus were obtained using standard methods. According to the manufacturer's instructions, cells were plated at 5 x 10 5 cells / ml were resuspended in BD Pharmingen FACS staining buffer, blocked with anti-mouse CD16 / 32 (BD Pharmingen), stained with the appropriate antibody mix, and stained with BD CYTOFIX TM fixed. The final cell pellet was resuspended in 0.5 mL of staining buffer and stained using BD FACSCALIBUR TM and BD CELLQUEST PRO TM software for analysis. All antibodies (BD Pharmingen) were prepared in substance diluent / mix and spiked to 0.5 mg / 10 5 final concentration of cells. The antibody mixture (A-D) used for bone marrow staining is as follows: A: anti-mouse IgM b -FITC, anti-mouse IgM a -PE, anti-mouse CD45R(B220)-APC; ...

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Abstract

Mice, tissues, cells, and genetic material are provided that comprise a humanized heavy chain immunoglobulin locus, a humanized light chain locus that expresses a universal light chain, and a gene encoding an ADAM6 or ortholog or homolog or functional fragment thereof. Mice are provided that express humanized heavy chains comprising human variable domains, and that express humanized light chains comprising human variable domains wherein the light chains are derived from no more than one, or no more than two, light chain V and J or rearranged V / J sequences. Fertile male mice that express antibodies with universal light chains and humanized heavy chains are provided. Methods and compositions for making bispecific binding proteins are provided.

Description

technical field [0001] Sequences and antibodies useful in making human immunoglobulin heavy chain variable domain encoding include bispecific antibodies and genetically modified mice, cells, embryos, tissues and isolated nucleic acids including bispecific antibodies comprising universal light chains. Compositions and methods comprising genetically modified mice having germline substitutions of endogenous mouse heavy chain variable loci comprising modified light chain loci expressing derived A light chain from no more than one or two different light chain V gene segments, wherein the mouse is further genetically modified in its germline such that male mice with these modifications are capable of breeding. The present application provides genetically modified mice expressing a universal light chain and a humanized heavy chain variable domain, wherein the mice comprise ADAM6 activity that is functional in male mice. Background technique [0002] The development of antibodies f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85A01K67/027C12N9/64C07K16/46
CPCC07K16/00C07K16/2833C07K16/40C12N9/6489C12N15/8509A01K2207/15A01K2217/072A01K2217/15A01K2227/105A01K2267/01C07K2317/21C07K2317/24C07K2317/515C07K2317/76C07K2317/92C07K2319/30C12N2800/204C12N2800/30A01K67/0278
Inventor J·麦克沃克L·麦克唐纳德S·史蒂文斯S·戴维斯D·R·巴克勒K·A·霍西阿瓦A·J·莫菲
Owner REGENERON PHARM INC
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