Method for the Generation of Genetically Modified Vertebrate Precursor Lymphocytes and Use Thereof for the Production of Heterologous Binding Proteins

Abstract

The present invention generally relates to the fields of genetic engineering and antibody production. In particular, it relates to the generation of genetically modified vertebrate precursor lymphocytes that have the potential to differentiate into more mature lymphoid lineage cells, and to the use thereof for the production of any heterologous antibody or binding protein.

1 . A method for the generation of vertebrate lymphocytes that can be used for the production of any heterologous antibody, antigen receptor, artificial binding protein, or functional fragment(s) thereof, comprising the steps of:
 (a) genetically modifying vertebrate precursor lymphocytes, which
 (i) are derived from primary lymphoid organs, and 
 (ii) have the potential to differentiate into mature lymphoid lineage cells, by introducing at least one exogenous genetic element encoding at least one heterologous antibody, antigen receptor, artificial binding protein, or functional fragment thereof; and 
   (b) effecting differentiation of said genetically modified precursor lymphocytes into mature lymphoid lineage cells either in vitro or in vivo, thereby generating lymphocytes capable of producing said heterologous antibody, antigen receptor, artificial binding protein or functional fragment(s) thereof.   
     
     
         2 . The method according to  claim 1 , further comprising the step of immortalizing the differentiated lymphocytes by:
 (a) fusing the same to myeloma cells for the generation of hybridoma cells;   (b) infecting the same with transforming viruses; or   (c) transfecting the same with a vector construct ensuring expression of at least one transforming oncogene;   thereby generating vertebrate lymphocytes capable of permanently producing said heterologous antibody, antigen receptor, artificial binding protein, or functional fragment(s) thereof.   
     
     
         3 . The method according to  claim 1 , wherein the vertebrate precursor lymphocytes are able to express at least one component of the lymphoid V(D)J recombination machinery and originate from jawed vertebrates comprising cartilaginous fish, bony fish, amphibians, reptilia, birds, mammals including pigs, sheep, cattle, horses and rodents including mice, rats, rabbits and guinea pigs, with murine precursor (pre) B lymphocytes from mice being preferred. 
     
     
         4 . The method according to  claim 1 , wherein said vertebrate precursor lymphocytes are deprived of their potential to express endogenous antibodies and/or antigen receptors or functional fragment(s) thereof, which is achieved by isolating/selecting vertebrate precursor lymphocytes being deficient in expressing endogenous immunoglobulins or fragments thereof, and/or by introducing into said vertebrate precursor lymphocytes at least one vector construct designed to functionally inactivate at least one allele of at least one genetic element, which is selected from the group consisting of:
 (a) the coding regions of the immunoglobulin heavy chain gene locus, including all or parts of the V, D, and J gene segments, and any of the coding regions for the constant region exons for μ, δ, γ, ε, and α heavy chains, with or without their membrane spanning exons;   (b) the coding regions of the immunoglobulin κ and/or λ light chain gene loci, including any of the V and J gene segment coding regions, as well as any of the constant region exons;   (c) the coding regions of the T cell receptor α, β, γ, and δ gene loci, including all or parts of the V, D and J gene segments, and any of the coding regions for the α, β, γ, and δ constant region exons;   (d) the cis-acting immunoglobulin heavy chain gene locus enhancer elements, including the heavy chain intron enhancer and the 3′α enhancer;   (e) the cis-acting immunoglobulin light chain gene locus enhancer elements, including the κ light chain intron enhancer (κiE), the 3′κ enhancer, and the λ2-4 and λ3-1 enhancers;   (f) the cis-acting T cell receptor gene loci enhancer elements, including the TCR α, β, γ, and δ enhancers;   (g) the trans-acting recombination activating genes, RAG-1 and RAG-2, including their promoter and enhancer elements, as well as their coding regions; and   (h) the trans-acting DNA repair genes essential for V(D)J recombination, including Ku70, Ku86, the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), DNA ligase IV, XRCC4 and Artemis, including their promoter and enhancer elements, as well as the coding regions of said genes.   
     
     
         5 . The method according to  claim 4 , wherein said vector constructs include gene targeting vectors comprising regions of DNA sequence homology to said at least one genetic element enabling for homologous recombination, preferably flanking a positive selection marker enabling selection of positive transfectants. 
     
     
         6 . The method according to  claim 5 , wherein said gene targeting vectors additionally comprise a pair of DNA recognition sequences for site-specific DNA recombination enzymes, flanking the positive selection marker, enabling deletion of said positive selection marker upon transfection and transient expression of nucleic acid sequences encoding at least one of the cognate recombinase enzymes. 
     
     
         7 . The method according to  claim 5 , wherein said gene targeting plasmid vectors additionally comprise a negative selection marker enabling selection against transfectants in which said gene targeting vectors are randomly integrated into the genome by non-homologous recombination. 
     
     
         8 . The method according to  claim 1 , wherein said at least one exogenous genetic element encoding a heterologous antibody, an artificial binding protein, an antigen receptor, or (a) functional fragment(s) thereof, is carried on a genetic construct selected from the group consisting of:
 (a) recombinant retroviral DNA constructs comprising promoter, enhancer and coding nucleic acid sequences operably linked to allow expression of at least one heterologous antibody, artificial binding protein, antigen receptor, or functional fragment thereof, being either wild-type or having (a) designed mutation(s) in the primary amino acid sequence(s) or being artificial;   (b) recombinant plasmid-based DNA constructs comprising promoter, enhancer and coding nucleic acid sequences operably linked to allow expression of at least one heterologous antibody, artificial binding protein, antigen receptor, or functional fragment thereof, being either wild-type or having (a) designed mutation(s) in the primary amino acid sequence(s) or being artificial;   (c) recombinant plasmid-based mini-immunoglobulin or T cell receptor gene loci with unrearranged V, D and J gene segments operably linked to allow V(D)J recombination and subsequent expression of at least one heterologous antibody or T cell receptor, or functional fragment thereof, being either wild-type or having (a) designed mutation(s) in the primary amino acid sequence(s);   (d) bacterial, yeast or vertebrate artificial chromosomes comprising parts or all of immunoglobulin or T cell receptor gene loci in germline configuration operably linked to allow V(D)J recombination and subsequent expression of at least one heterologous antibody or T cell receptor, or functional fragment thereof, being either wild-type or having (a) designed mutation(s) in the primary amino acid sequence(s);   (e) bacterial, yeast or vertebrate artificial chromosomes comprising parts or all of at least one heterologous immunoglobulin or T cell receptor gene locus in modified arrangement designed to allow (D)J recombination and subsequent expression of at least one heterologous antibody or T cell receptor, or functional fragment thereof, being either wild-type or having (a) designed mutation(s) in the primary amino acid sequence(s);   (f) trans-chromosome elements which are fragments of heterologous chromosomes harbouring parts or all of immunoglobulin or T cell receptor gene loci in germline configuration allowing V(D)J recombination and subsequent expression of at least one heterologous antibody or T cell receptor, being wild-type with respect to the primary amino acid sequence(s).   
     
     
         9 . The method according to  claim 1 , wherein the at least one exogenous genetic element encodes a native or modified human antibody, a human binding protein, a human antigen receptor, or (a) functional fragment(s) thereof. 
     
     
         10 . The method according to  claim 1 , wherein the at least one exogenous genetic element encodes a heterologous or artificial receptor capable of undergoing affinity maturation of the binding region. 
     
     
         11 . The method according to  claim 1 , wherein the differentiation of vertebrate precursor lymphocytes is effected in vitro by:
 (a) arresting proliferation of said vertebrate precursor lymphocytes and inducing differentiation into mature lymphocyte lineage cells by cultivating in the absence of any precursor lymphocyte growth factor; and   (b) inducing terminal lymphocyte differentiation by further cultivating said cells in the presence of at least one of the following components selected from:
 (i) soluble T cell related stimulating factors, comprising interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin-10, interleukin-13, TGF-β, and IFN-γ; 
 (ii) factors activating co-stimulatory receptors of B cells, comprising agonistic antibodies or active, recombinant ligands specific for CD40, B7-1 (CD80), B7-2 (CD86), complement receptors 1 (CD35) and 2 (CD21), LFA-1 (CD11 a), LFA-3 (CD58), CD19, CD20, CD30, CD32, CD37, CD38, CD70, CD71, Iga (CD79α), Igβ(3 (CD79β), TAPA-1 (CD81), Fas (CD95), TNF-receptor1 (p55, CD120α), TNF-receptor2 (p75, CD120β), Ox-40 (CD134), and lymphotoxin-/3 receptor; and 
 (iii) B cell mitogenic factors, T cell independent antigens of type 1, and other polyclonal activators, including lipoplysaccharide (LPS), lipoproteins from gram negative bacteria, polyanions, poly-dIdC, pokeweed mitogen (PWM), and anti-immunoglobulin reagents; 
   and combinations thereof.   
     
     
         12 . The method according to  claim 1 , wherein said differentiation in vivo is effected upon transplantation of said genetically modified precursor lymphocytes into a suitable vertebrate host. 
     
     
         13 . The method according to  claim 12 , wherein said lymphocytes are co-transplanted into said host with naive or antigen primed T helper lymphocytes. 
     
     
         14 . The method according to  claim 12 , wherein said differentiation in vivo is followed by immunization of said host with at least one desired immunogenic compound or composition. 
     
     
         15 . The method according to  claim 12 , wherein said vertebrate host is a compatible host being deficient with respect to the generation of endogenous B cells, T cells, and/or NK (natural killer) cells, or a combination thereof. 
     
     
         16 . The method according to  claim 12 , wherein the vertebrate host is selected from jawed vertebrates comprising cartilaginous fish, bony fish, amphibians, reptilia, birds, and mammals including pigs, sheep, cattle, horses and rodents including mice, rats, rabbits and guinea pigs, with mice being the preferred host species. 
     
     
         17 . A method comprising the steps of: (i) obtaining genetically modified and differentiated vertebrate lymphocytes by the method according to  claim 1 , and (ii) using the lymphocytes to produce a heterologous antibody, artificial binding protein, antigen receptor, or functional fragment(s) thereof. 
     
     
         18 . The method according to  claim 17 , wherein the method according to  claim 1  is followed by the steps of:
 (a) isolating from said differentiated lymphocytes the at least one exogenous genetic element, and 
 (b) placing said genetic element(s) in a context enabling production of said at least one heterologous antibody, antigen receptor, artificial binding protein, or functional fragment(s) thereof. 
 
     
     
         19 . The method according to  claim 17 , wherein said heterologous antibody, artificial binding protein, antigen receptor, or functional fragment(s) thereof, displays one unique specificity and is therefore monoclonal. 
     
     
         20 . The method according to  claim 17 , wherein said heterologous antibody, artificial binding protein, antigen receptor, or functional fragment(s) thereof, displays more than one unique specificity and is therefore polyclonal. 
     
     
         21 . The method according to  claim 17 , wherein said heterologous antibody, artificial binding protein, antigen receptor, or functional fragment(s) thereof, completely or partially resembles a human antibody, antigen receptor, or binding protein, as can be assembled on the basis of the human genetic repertoire or parts thereof. 
     
     
         22 . The method according to  claim 17 , wherein the heterologous antibody, artificial binding protein, antigen receptor, or functional fragment(s) thereof to be produced is selected from the group consisting of:
 (a) antibodies being either membrane bound or secreted, and consisting of both heterologous heavy and light chain polypeptides in the stoichiometric composition found in natural antibodies and consisting of any of the known heavy (μ, δ, γ, α, ε) and/or light (κ and λ) chain isotypes;   (b) antibodies with combinations of heavy and light chain polypeptides being completely human with respect to the primary amino acid sequence;   (c) hybrid antibodies containing heterologous heavy or light chain polypeptides from different vertebrate species;   (d) secreted Fab, scFv and F(ab′) 2  antibody fragments being either completely or partially heterologous;   (e) fragments of antibodies covalently coupled via linker peptides, resulting in bispecific or multispecific antibody fragments;   (f) T cell receptors of the α, β, γ, and δ isotype, antigen receptors, and other binding proteins with structural resemblance to proteins of the immunoglobulin superfamily;   and functional fragments thereof.   
     
     
         23 . Genetically modified vertebrate precursor lymphocytes and more mature lymphoid lineage cells derived therefrom, obtainable by a method according to  claim 1 . 
     
     
         24 . Immortalized cells producing heterologous antibodies, artificial binding proteins, antigen receptors, or functional fragments thereof, obtainable according to  claim 2 . 
     
     
         25 . Vector constructs for use in a method according to  claim 4 . 
     
     
         26 . Genetic construct for use in the method according to  claim 8 . 
     
     
         27 . Pharmaceutical or diagnostic preparation, comprising at least one antibody, artificial binding protein, antigen receptor, or functional fragment thereof, obtained by a method according to  claim 17 , displaying either wild-type immune effector functions, or modified or artificial effector functions not derivable from germline encoded heterologous immunoglobulins or antigen receptors.