Patent Application
20100122358
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The present invention relates to heavy (H)-chain-only antibodies, for example produced in light (L)-chain loci-deficient mice, production of the antibodies, and uses thereof. The present invention further relates to a method for the production of a heavy (H)-chain-only immunoglobulin (Ig) A binding molecule in a light (L)-chain loci-deficient mouse, the binding molecule per se, and uses thereof.
Most natural antibodies or immunoglobulins (Ig's) typically comprise two heavy (H-) chains and two light (L-) chains. The H-chains are joined to each other by disulphide bonds located near flexible hinge domains, and each L-chain is associated with the N-terminal part of the C-chain by a disulphide bond. Each L-chain has a variable (VL) and a constant (CL) domain, while each H-chain comprises a variable domain (VH), a first constant domain (CH1), a hinge domain and two or three further constant domains (CH2, CH3 and optionally CH4). In normal dimeric antibodies, interaction of the VH and VL domains forms an antigen binding region, although binding is facilitated by the CH1 domain and parts of the CL domain.
Several different classes of natural Ig have been identified. These classes differ in the constant domains of their H-chains, which in turn affects the function of the Ig. In mammals, the five type of Ig are IgA, IgD, IgE, IgG and IgM. Humans and mice have four IgG subtypes, and humans have two IgA subtypes. IgA comprises three CH domains encoded by Cα gene segments, including αCH1 (also termed Cα1), αCH2 (also termed Cα2) and αCH3 (also termed Cα3) genes encoding αCH1, αCH2 and αCH3 domains, respectively. IgA plays a central role in mucosal immunity, which is established after release of IgA from a plasma cell and transport to the mucosal epithelial cell layer. In this layer, polymeric IgA is bound to a polymeric Ig receptor, which, after cleavage, provides the secretory component important for stabilization and conferring resistance to attack by proteases (reviewed in ref. 81). Secretory IgA is dimeric, containing two H2L2 units joined by one J chain (82), and is generally abundant in secretions such as milk and colostrum. Serum IgA is present at lower levels in the mouse, mainly in a dimeric form, whereas in humans it is more highly expressed but monomeric.
IgD comprises CH domains encoded by Cδ gene segments, is monomeric, and functions as an antigen receptor on B cells, the cells responsible for producing antibodies. IgE has CH domains encoded by Cε gene segments, is also monomeric, and binds to allergens and receptors on mast cells, which triggers the release of cytokines and histamine (allergy response). IgG comprises CH domains encoded by Cγ gene segments, is monomeric, and provides most of the antibody-based (humoral) response against pathogens. Finally, IgM has CH domains encoded by Cμ gene segments, is pentameric, and is expressed on the surface of B cells and also in a secreted form. Secreted IgM has a role in eliminating pathogens in the early stages of B cell-mediated immunity.
In the mammalian immune system, DNA recombination and surface IgM expression are required for B-lymphocyte development. In bone marrow B cells, D to JH rearrangement is completed at the pre B1 stage. This is followed by VH to DJH rearrangement in large pre B2 cells, and VL to JL in small pre B2 cells, indicating sequential differentiation events (1-3). At the pre B2 cell stage, replacement of surface-expressed surrogate L-chain by kappa (κ) or lambda (λ) L-chain initiates the process of antibody maturation, which is accompanied by cellular migration and class-switching. Mature B cells undergo further selection and can differentiate into antibody secreting plasma cells or memory B cells bearing different isotypes (IgG, IgA or IgE). Checkpoints during the progression of these regular events ensure that only cells with productive rearrangements advance in differentiation (4). The formation of the B cell receptor (BCR) and its associated chains are regarded as essential to allowing normal B cell development (5). This has been confirmed in mice lacking the H-, L-, Igα or Igβ polypeptide of the BCR (6-8).
Normal Ig expression in B cells involves an ordered succession of gene rearrangements. Exons encoding variable regions of H-chains are constructed in vivo by assembly of VH, diversity (D) and joining (JH) segments, while for L-chains V and JL segments are assembled. During B-cell development, the genes involved in recombinase activity controlling V(D)J rearrangements are specifically expressed at the pre-B cell stage. The rearranged VDJ region is initially transcribed in association with the Cμ gene segment, leading to the synthesis of an IgM H-chain. Subsequently, by a process called switch recombination, the Cμ gene segment is deleted and the downstream Cδ gene segment is used to synthesise an IgD H-chain. The process of isotypic switching continues by bringing further downstream CH (γ, α or ε) gene segments close to the VDJ exon. Switch regions within each of the gene segments are required for switch recombination. In mice, the H-chain gene segment order is 5′-D-JH-Cμ-Cδ-Cγ3-Cγ1-Cγ2b-Cγ2a-Cε-Cα-3′. The human H-chain gene segment order is 5′-D-JH-Cμ-Cδ-Cγ3-Cγ1-Cψε2-Cα1-Cγ2-Cγ4-Cε1-Cα2-3′.
In Tylopoda or camelids (dromedaries, camels and llamas), a major type of Ig, composed solely of paired H-chains (9), is produced in addition to conventional antibodies of paired H- and L-chains (10). The secreted homodimeric H-chain-only antibodies found in these animals use specific VH (VHH) and γ genes which result in a smaller than conventional H-chain, lacking the C(constant)H1 domain. Interestingly, H-chain antibodies are also present in some primitive fish; e.g. the new antigen receptor (NAR) in the nurse shark and the specialized H-chain (COS5) in raffish (11, 12). Again these H-chain Igs lack the CH1-type domain. However, evolutionary analysis has shown that their genes emerged and evolved independently, whereas H-chain genes in camelids evolved from pre-existing genes used for conventional heteromeric antibodies (13). H-chain antibodies can also be found in humans with Heavy Chain Disease (HCD) where the H-chain-only Ig has part of the VH and/or CH1 domain removed (14).
It has also been shown that Tylopoda or camelids (camels, dromedaries and llamas), and recently by us in mice, that H-chain-only IgG antibodies are expressed when the γCH1 exon is removed by splicing of the RNA transcript or DNA deletion, respectively (9, 49, 86). The loss of this exon fits with its putative function of providing a disulphide linkage to the L-chain. Parallels have been drawn to the expression of H-chain-only antibodies in cartilaginous fish, which also lack CH1 or a CH1-type domain (87, 12). These single chains are comprised of a flexible assembly of 3-5 Cμ domains, and are part of a large assortment of isotypes of different lengths and function found in lower vertebrates, possibly arising by differential splicing to overcome proteolysis (88).
The synthesis of abnormal Ig has been reported in humans with various immunoproliferative disorders. In the case of heavy-chain disease (HCD) where H-chain-only Ig proteins are produced, lymphoid proliferation is associated with pathological and clinical features. One form of HCD, αHCD, is prevalent in developing countries (84), and accompanied by rapid expansion of B cells producing truncated α H-chain (85). Characterisation of a range of HCD Ig's reveals that most of the abnormal proteins have an isotype not from the most 3′ CH gene segments (such as Cγ2-Cγ4-Cα2) but from the most 5′ gene segments (such as Cμ, Cγ3, Cγ1 or Cα1). In humans, it is considered that the switch regions of the most 5′ CH gene segments are more susceptible to abnormal deletions than switch regions of the 3′ CH gene segments (43).
Intracellular transport of Ig is dependent on its correct folding and assembly in the endoplasmic reticulum, where single H-chain is chaperoned by non-covalent association with the H-chain binding protein BiP or grp78 (15). The BiP/H-chain complex is formed by virtue of the KDEL sequence at the carboxy terminus of BiP (16) and the CH1 domain of the H-chain. When L-chain displaces BiP Ig can go to the cell surface or be secreted. If CH1, or part of VH, is missing L-chain is no longer required to replace BiP and the H-chain can travel unhindered to the cell surface and be secreted as seen in animals that make H-chain-only antibodies and in HCD.
The present invention arises from the surprising finding (see examples below) that diverse H-chain-only IgG without CH1 is found in the serum of mice deficient in L-chain but without further genetic manipulation, despite compromised B cell development in these mice. We have found that H-chain-only IgGs are produced from naturally- or endogenously-produced transcripts lacking the CH1 exon.
The invention relates to mice having functionally silenced endogenous lambda (λ) and kappa (κ) L-chain loci, comprising antibody-producing cells in which the CH1 domain is functionally silenced, either via spontaneous processes in somatic antibody-producing cells or due to germline deletion of the CH1 domain. Mice of the invention are capable of producing H-chain-only antibody lacking a functional CH1 domain; transgenic human heavy-chain-only antibodies lacking a functional CH1 domain can be produced following insertion into the mouse of an artificial locus with human heavy chain V, D and J segments and a constant region, which is preferably a modified constant region with alterations in, around or upstream of a CH1 domain and/or removal of a CH1 domain. According to a first aspect of the present invention, there is provided a mouse having functionally silenced endogenous lambda (λ) and kappa (κ) L-chain loci, in which the mouse comprises an antibody-producing cell that produces a H-chain-only antibody lacking a functional CH1 domain following in vivo functional silencing of a gene encoding the CH1 domain.
Unlike prior art mice used in the production of H-chain-only antibodies (see for example WO2006/008548), in some embodiments the invention does not require artificial genetic manipulation to functionally silence (for example, by deletion or disruption) the CH1 domain within an H-chain locus prior to insertion of the locus into a mouse for the production of H-chain-only antibodies. Rather, in some embodiments, the invention utilises previously undescribed natural, spontaneous processes in the L-chain deficient mice to functionally silence the CH1 domain.
The mouse having functionally silenced endogenous lambda and kappa L-chain loci may, for example, be made as disclosed in WO03/000737, which is hereby incorporated by reference in its entirety. In WO03/000737 functional silencing of the Igκ locus was achieved by insertion of neo into Cκ (46); functional silencing of the λ locus was by a Cre-IoxP mediated deletion of ˜120 kb encompassing all Cλ genes.
The mouse may also have a functionally silenced endogenous heavy chain locus, for example produced as disclosed in WO04/076618, which is hereby incorporated by reference in its entirety. In WO04/076618 functional silencing of the endogenous heavy chain constant region locus was achieved by Cre-IoxP mediated deletion of the heavy chain constant region genes.
Preferably the mouse is capable of expressing pre-BCR and/or surface display of an endogenous or exogenous IgM.
The mouse of the invention may additionally comprise a nucleic acid construct integrated into the endogenous mouse genome, in which the nucleic acid construct comprises non-murine heavy chain genes from which the H-chain-only antibody is produced.
The non-murine heavy chain genes may be from other vertebrates including mammals such as from a rat or particularly from a human.
One example of a suitable construct comprising human heavy chain genes is the IgH YAC construct disclosed in WO2004/049794 and/or reference 80, which are hereby incorporated by reference in their entirety.
The nucleic acid construct may additionally comprise one or more mouse CH genes, including a CH1 gene. This will allow natural processing mechanisms to functionally silence the CH1 gene to produce a H-chain-only antibody.
The nucleic acid construct may exclude a functional human CH1 gene.
In vivo functional silencing of the CH1 domain gene according to the invention may be achieved by class switch recombination. For reasons elaborated in the specific embodiments, it is understood that the natural mechanism of in vivo functional silencing of CH1 domain genes in L-chain deficient mice is class switch recombination. Class switch recombination has been described in the prior art, for example see references 25, 33 and 52, which are hereby incorporated by reference in their entirety.
According to another aspect of the invention there is provided a transgenic mouse of the invention having a nucleic acid construct integrated in the endogenous mouse genome, in which the nucleic acid construct comprises non-murine vertebrate (for example, human) V, D and J region genes and in which the mouse produces a mouse-non-murine vertebrate (such as mouse-human) chimeric H-chain-only antibody comprising non-murine vertebrate (for example, human) V, D and J domains and one or more mouse CH domains excluding a functional CH1 domain.
In this aspect of the invention, the mouse may recognise mouse CH genes and associated regulatory switch recombination sequences (see below) to allow in vivo functional silencing of the CH1 domain and thereby formation of an H-chain-only antibody.
The non-murine vertebrate (for example, human) V, D and J region genes in the construct may be in non-murine vertebrate (for example, human) or murine (mouse) germline configuration. Non-murine vertebrate (for example, human) germline configuration will be suitable for achieving similar selection and maturation (recombination) events in the V, D and J regions to those found in the non-murine vertebrate (for example, human). Re-positioning of the human V, D and J region genes in the construct to mirror mouse germline configuration will allow efficient selection and maturation (recombination) events of the non-murine vertebrate (for example, human) V, D and J regions within the mouse.
The non-murine vertebrate (for example, human) V, D and J domains of the antibody in this aspect of the invention preferably result from recombination of the non-murine vertebrate (for example, human) V, D and J region genes. The process of somatic hypermutation will allow development of a diverse H-chain-only antibody repertoire.
The nucleic acid construct in the mouse may be integrated upstream of endogenous mouse CH region genes. This will allow in vivo functional silencing of the endogenous CH1 domain gene and formation of the H-chain-only antibody. Alternatively, the nucleic acid construct in the mouse may comprise one or more mouse CH region genes including a CH1 domain gene. Here, in vivo functional silencing of the introduced construct CH1 domain gene allows formation of the H-chain-only antibody. In both cases, the CH1 domain gene may be functionally silenced by class switch recombination.
According to a further aspect of the invention there is provided a transgenic mouse having a nucleic acid construct integrated in the endogenous mouse genome, in which the nucleic acid construct comprises:
(i) non-murine vertebrate (for example, human) heavy chain V, D, J and C genes, for example in non-murine vertebrate (for example, human) or mouse germline configuration, including a non-murine vertebrate (for example, human) CH1 gene; and
(ii) class switch recombination sequences upstream of the non-murine vertebrate (for example, human) CH1 gene.
The class switch recombination sequences may facilitate class switch recombination-mediated functional silencing of the non-murine vertebrate (for example, human) CH1 gene in vivo, thereby allowing production of a non-murine vertebrate (for example, human) H-chain-only antibody in the mouse.
Class switch recombination sequences for use in the invention, for example mouse class switch recombination sequences, are described in the specific embodiments below and are also as known in the art (see for example references 25, 33 and 52, incorporated herein by reference in their entirety). These sequences facilitate the class switch recombination process to allow functional silencing of the CH1 gene by deletion.
The mouse of the invention in one aspect lacks or is deficient in B cell receptor (BCR)-expressing B cells.
The mouse in one aspect does not exhibit lymphoproliferation as seen in human H chain disease (HCD; see reference 14).
The mouse of the invention may be inbred through two or more generations to increase production of the H-chain-only antibodies. We have found in particular that mice with functionally silenced endogenous lambda (λ) and kappa (κ) L-chain loci when bred through successive generations increase the serum level of H-chain-only antibody production, presumably due to selection of antibody transcripts with functionally silenced CH1 domains which produce the expressed antibodies.
In another aspect of the invention there is provided an isolated nucleic acid (for example, a vector such as a BAC, YAC or artificial chromosome) comprising a construct or an antibody as described herein.
According to another aspect of the invention there is provided a host cell comprising the nucleic acid as defined above.
A further aspect of the invention is use of a mouse of the instant invention in the production of an H-chain-only antibody lacking a functional CH1 domain following in vivo functional silencing of a gene encoding the CH1 domain.
Also provided is a method for obtaining an H-chain-only antibody from a mouse, comprising the steps of:
(i) producing a mouse with functionally silenced endogenous lambda and kappa L-chain loci;
(ii) allowing formation in the mouse of an H-chain-only antibody lacking a functional CH1 domain following in vivo functional silencing of a gene encoding the CH1 domain; and
(iii) obtaining the H-chain-only antibody from mouse serum.
A specific embodiment of this aspect is a method for obtaining an IgG-type H-chain-only antibody from a mouse, comprising the steps of:
(i) producing a mouse with functionally silenced endogenous lambda and kappa L-chain loci using the method described in WO03/000737;
(ii) allowing in vivo functional silencing of a gene encoding a CH1 domain in the mouse;
(iii) forming an IgG-type H-chain-only antibody lacking a functional CH1 domain; and
(iv) obtaining the IgG-type H-chain-only antibody from mouse serum.
According to another aspect of the invention there is provided a method for isolating an antibody-producing cell which produces an antigen-specific H-chain-only antibody (for example, an IgG-type H-chain-only antibody), comprising the steps of:
(i) obtaining a mouse with functionally silenced endogenous lambda and kappa L-chain loci;
(ii) immunizing the mouse with an antigen;
(iii) selecting for a cell producing an antigen-specific H-chain-only antibody lacking a functional CH1 domain following in vivo functional silencing of a gene encoding the CH1 domain; and
(iv) isolating the cell selected in step (iii).
In this aspect of the invention, the mouse may be produced using the method described in WO03/000737.
Selection and isolation of the cell may employ flow-cytometry, for example for the identification and isolation of B220int/+, syndecan+ spleen-derived plasma cells in which an antigen-specific H-chain-only antibody lacking a functional CH1 domain is produced.
In an alternative aspect, peritoneal cells are selected and isolated.
Another embodiment of this aspect of the invention is a method for isolating an antibody-producing cell which produces an antigen-specific IgG-type H-chain-only antibody, comprising the steps of:
(i) obtaining a mouse with functionally silenced endogenous lambda and kappa L-chain loci using the method described in WO03/000737;
(ii) immunizing the mouse with an antigen;
(iii) isolating sub-populations of cells from secondary lymphoid organs or bone marrow in the mouse;
(iv) screening the sub-populations of cells isolated in step (iii) by RT-PCT using JH to γCH2 amplifications to detect mutant γ H chain transcripts in which the CH1 exon has been deleted; and
(v) selecting those sub-populations of cells screened in step (iv) which have mutant γ H chain transcripts; and
(vi) isolated cells selected in step (v).
The antibody-producing cell of this aspect of the invention may be isolated from a secondary lymphoid organ. For example, the secondary lymphoid organ may be a non-splenic organ, for example any of the group consisting of: lymph node, tonsil, and mucosa-associated lymphoid tissue (MALT), including gut-associated lymphoid tissue (GALT), bronchus-associated lymphoid tissue (BALT), nose-associated lymphoid tissue (NALT), larynx-associated lymphoid tissue (LALT), skin-associated lymphoid tissue (SALT), vascular-associated lymphoid tissue (VALT), and/or conjunctiva-associated lymphoid tissue (CALT). In one embodiment, the antibody-producing cell is a peritoneal cell.
In the methods of the invention, the produced H-chain-only antibody may be non-murine vertebrate (for example, human) or a mouse-non-murine vertebrate (for example, human) chimera. Where a mouse-non-murine vertebrate chimeric (for example, a mouse-human chimeric) H-chain-only antibody is produced, the antigen-specificity-determining regions (defined by the VDJ domains) may be non-murine vertebrate (for example, human), with the C regions being of mouse origin.
Also provided is an isolated antibody-producing cell obtainable using the method of the invention.
Further provided according to the invention is a hybridoma obtainable by fusion of an antibody-producing cell as defined herein with a B-cell tumor line cell. We have found (see Example 2) that H-chain-only antibody production is not dependent on the presence of a mouse spleen, so in certain embodiments of the invention the antibody-producing cell used to form the hybridoma is a non-splenic secondary lymphoid organ cell (see above). Well known methods of generating and selecting single clone hybridomas for the production of monoclonal antibodies may be adapted for use in the present invention.
The invention in another aspect provides an H-chain-only antibody lacking a functional CH1 domain following in vivo functional silencing of a gene encoding the CH1 domain, or a fragment of the antibody.
The antibody of the invention may be produced in mouse having functionally silenced endogenous lambda and kappa L-chain loci.
The antibody of the invention may be in an isolated and purified form. The antibody may be isolated and/or characterised using methods well known in the art. Once characterised, the antibody or the fragment thereof may be manufactured using recombinant or synthetic methods, also well known in the art. For applicable prior art methods, see references listed below.
The antibody may be modified to increase solubility, for example by genetic engineering of one or more genes encoding the antibody.
The antibody of the invention may be specific to an antigen. The antibody may be engineered to be a bi- or multi-valent antibody with one or more specificities.
The antibody of the invention may be a monoclonal antibody.
The antibody of the invention may be an IgG-like antibody or an IgM-like antibody (as exemplified below).
In one aspect of the invention, the H-chain-only antibody has the structure VHDJH-hinge-CH2-CH3. Each domain of the antibody may be of non-murine vertebrate (for example, human) or of mouse origin, or the antibody may be chimeric (for example where the VHDJH part of the structure is non-murine vertebrate, such as human, and the hinge-CH2-CH3 part of the structure is murine).
The antibody of the invention in preferred embodiments lacks endogenous gross alteration of the VH regions as seen in human HCD (see reference 14).
The antibody of the invention in certain embodiments does not include one or more or all camelid VHH-specific mutations found in VH to VHH substitutions in framework 2 (i.e. Val37Phe, Gly44Glu, Leu45Arg and Trp47Gly). As shown below, such mutations are not required for production of H-chain-only antibodies in L-chain-deficient mice.
The antibody of the invention may be used as a diagnostic, prognostic or therapeutic imaging agent. The antibody may additionally or alternatively be used as an intracellular binding agent, or an abzyme.
The antibody of the invention may be for use as a medicament in the treatment of a disease.
The antibody of the invention may be for use in the manufacture of a medicament in the treatment of a disease.
Also provided is a medicament comprising an antibody of the invention. The medicament will typically be formulated using well-known methods prior to administration into a patient.
In a further aspect of the invention there is provided a method of treating a disease, comprising the step of administering a medicament of the invention to a patient in need of same.
Diseases which are susceptible to treatment using an antibody include: wound healing, cell proliferative disorders, including neoplasm, melanoma, lung, colorectal, osteosarcoma, rectal, ovarian, sarcoma, cervical, oesophageal, breast, pancreas, bladder, head and neck and other solid tumors; myeloproliferative disorders, such as leukemia, non-Hodgkin lymphoma, leukopenia, thrombocytopenia, angiogenesis disorder, Kaposis' sarcoma; autoimmune/inflammatory disorders, including allergy, inflammatory bowel disease, arthritis, psoriasis and respiratory tract inflammation, asthma, immunodisorders and organ transplant rejection; cardiovascular and vascular disorders, including hypertension, oedema, angina, atherosclerosis, thrombosis, sepsis, shock, reperfusion injury, and ischemia; neurological disorders including central nervous system disease, Alzheimer's disease, brain injury, amyotrophic lateral sclerosis, and pain; developmental disorders; metabolic disorders including diabetes mellitus, osteoporosis, and obesity, AIDS and renal disease; infections including viral infection, bacterial infection, fungal infection and parasitic infection, pathological conditions associated with the placenta and other pathological conditions.
As used herein, the term “antibody” refers to both a naturally produced antibody which may be generated in response to an antigen and also where appropriate to a synthetic binding molecule which mimics the binding ability of a natural antibody, for example with modified binding or other pharmacological properties. Where appropriate, the term also encompasses antibody fragments, for example antigen-binding or effector antibody fragments.
Production of the H-chain only antibody according the invention may include expression from an antibody-producing cell, either by expression on the cell surface or by secretion (i.e. release of antibody from the cell).
As used herein, the term “in vivo” refers to an endogenous or a natural (non-engineered) process. The endogenous or natural process occurs spontaneously.
The term “mouse” used herein encompasses in further aspects of the invention other vertebrates such as mammals, preferably non-human mammals such as other rodents including rats.
As used herein, a non-murine vertebrate includes mammals such as a rat and a human, particularly a human.
The present invention further relates to a new type of H-chain-only binding molecule surprisingly found in L-chain deficient mice.
According to an aspect of the present invention, there is provided a method for producing an H-chain-only IgA binding molecule in a mouse, comprising the steps of:
(i) obtaining an L-chain deficient mouse with functionally silenced endogenous lambda and kappa L-chain loci; and
(ii) allowing formation in the L-chain deficient mouse of an H-chain-only IgA binding molecule lacking a functional αCH1 domain.
There are no published examples of the occurrence H-chain-only IgA binding molecules in healthy animals, only in humans with αHCD. Thus H-chain-only IgA have not been reported in camelids, which produce H-chain-only IgG, nor in elasmobranchs (sharks, skates and rays), where H-chain-only antibodies can comprise a variable number of Cμ domains (50, 88). The production of H-chain only IgA binding molecules according to the invention is also unexpected given the 3′ downstream location of the Cα gene segment compared with other Ig isotypes in mice. The invention allows production of H-chain-only IgA binding molecules (of murine or other origin) in a mouse, for example in stable and in relatively high amounts.
Furthermore, unlike prior art mice suggested for use in the production of H-chain-only antibodies (see for example WO2006/008548), this aspect of the invention does not require artificial genetic manipulation to functionally silence (for example, by deletion) the αCH1 domain within an αH-chain locus prior to insertion of the locus into a mouse for the production of H-chain-only IgA binding molecules.
The L-chain deficient mouse used in the method is in one aspect relatively healthy compared with a corresponding mouse without functionally silenced endogenous lambda and kappa L-chain loci, when kept under the same conditions (for example, pathogen-free conditions). The L-deficient mouse preferably does not show equivalent pathological and/or clinical symptoms seen in humans with αHCD (see ref. 43). For example, the L-deficient mouse may not exhibit lymphoproliferation.
The mouse having functionally silenced endogenous lambda and kappa L-chain loci may, for example, be made as described in WO03/000737, which is hereby incorporated by reference in its entirety. In WO03/000737 functional silencing of the Igκ locus was achieved by insertion of neo into Cκ((46); functional silencing of the λ locus was by a Cre-IoxP mediated deletion of ˜120 kb encompassing all Cλ genes.
The method of the invention may comprise a further step (iii) of isolating the H-chain-only IgA binding molecule.
In the method, the H-chain-only IgA binding molecule may be formed following in vivo functional silencing of a gene encoding the αCH1 domain. The H-chain-only IgA binding molecule may be formed following in vivo deletion of all or a part of the gene encoding the αCH1 domain. All or a part of the gene encoding the αCH1 domain may be deleted in vivo by imprecise class-switch recombination. Additionally or alternatively, all or a part of the gene encoding the αCH1 domain may be deleted in vivo due to one or more point mutations, out of frame reading, an incorrect stop codon and/or a splice site alteration.
In vivo deletion of all or a part of the gene encoding the αCH1 domain in one aspect of the invention is not accompanied by DNA insertion. In this aspect, the deletion mechanism is distinct from H-chain only antibody formation in αHCD where in-frame DNA insertions have been detected (43).
The H-chain-only IgA binding molecule may be produced in the L-chain deficient mouse at a level 0.2-2 times, for example about 0.25, 0.5, 1.0, 1.25, 1.5 or 1.75 times, that of a normal IgA antibody produced in a corresponding mouse without functionally silenced endogenous lambda and kappa L-chain loci. The level of production of the H-chain-only IgA binding molecule in the L-chain deficient mice is surprisingly high, as demonstrated in the specific embodiments below.
The L-chain deficient mouse may be at least 2.5 months old, for example at least 3 to 14 months old, such as about 5, 6, 9, 11, 12, 13 or 14 months old. We have found that generally older mice produce higher levels of H-chain-only IgA binding molecule.
The H-chain-only IgA binding molecule may be produced in a bone marrow cell, a mucosal cell (for example from a lamina propria or epithelial layer) and/or a spleen lymphocyte cell of the mouse. For example, the molecule may be produced in a spleen lymphocyte IgA+B220+ cell.
The H-chain-only IgA binding molecule produced according to the method may comprise a functional VH domain. In an aspect of the invention, the H-chain-only IgA binding molecule does not have a deletion and/or an insertion in any VH domains (as found in αHCD H-chain-only IgA antibodies).
The H-chain-only IgA binding molecule produced according to the method may comprise a functional αCH2 domain and/or a functional αCH3 domain.
The H-chain-only IgA binding molecule produced according to the method may comprise functional D and JH domains.
The H-chain-only IgA binding molecule produced according to the method in one aspect has functional domains in the following order: VH-D-JHαCH2-αCH3.
The H-chain-only IgA binding molecule produced according to the method may be a monomer. The monomer may have a single antigen binding site.
Alternatively, the H-chain-only IgA binding molecule produced according to the method may be a multimer for example a dimer or a tetramer. Each H-chain-only IgA binding molecule of the multimer may have a single antigen binding site. The multimer may thus have binding sites for more than one antigen.
The H-chain-only IgA binding molecule of the multimer may be associated with one or more J chains.
The H-chain-only IgA binding molecule produced according to the method may be a non-murine vertebrate binding molecule or a mouse-non-murine vertebrate chimeric binding molecule. The non-murine vertebrate may be a human.
The L-chain deficient mouse used in the method may additionally have all or part of its endogenous H-chain locus functionally silenced (for example produced as disclosed in WO04/076618, which is hereby incorporated by reference in its entirety). In WO04/076618 functional silencing of the endogenous heavy chain constant region locus was achieved by Cre-IoxP mediated deletion of the heavy chain constant region genes.
Preferably the mouse is capable of expressing preBCR and/or surface display of an endogenous or exogenous IgM.
The L-chain deficient mouse used in the method may additionally comprise a nucleic acid construct integrated into the endogenous mouse genome, in which the nucleic acid construct comprises non-murine H-chain genes from which the H-chain-only IgA binding molecule is produced. The non-murine H-chain genes may be from other vertebrates including mammals such as from a rat or particularly from a human. One example of a suitable construct comprising H-chain genes, optionally with modification as suggested herein, is the IgH YAC construct disclosed in WO2004/049794, which is hereby incorporated by reference in its entirety.
The invention accordingly encompasses a method of producing a human H-chain-only IgA molecule in the L-chain deficient mouse.
In one embodiment there is provided a method for obtaining an H-chain-only IgA binding molecule from a mouse, comprising the steps of:
(i) producing a mouse with functionally silenced endogenous lambda and kappa L-chain loci using the method described in WO03/000737;
(ii) allowing in vivo functional silencing of a gene encoding a αCH1 domain in the mouse;
(iii) forming an H-chain-only IgA binding molecule lacking a functional αCH1 domain; and
(iv) obtaining the H-chain-only IgA binding molecule from mouse serum, milk and/or saliva.
The nucleic acid construct may additionally comprise one or more mouse CH genes, including an αCH1 gene. This will allow natural processing mechanisms to functionally silence the αCH1 gene to produce an H-chain-only binding molecule. The nucleic acid construct may exclude a functional human αCH1 gene.
The L-chain deficient mouse used in the method may have integrated into its genome a nucleic acid construct comprising non-murine vertebrate V, D and J region genes and in which the mouse produces a mouse-non-murine vertebrate chimeric H-chain-only IgA binding molecule having non-murine vertebrate VH, D and JH domains and one or more mouse αCH domains other than a functional αCH1 domain.
In this aspect of the invention, the mouse may recognise mouse CH genes and associated regulatory switch recombination sequences (see below) to allow in vivo functional silencing of the CH1 domain and thereby formation of an H-chain-only antibody.
The non-murine vertebrate (for example, human) V, D and J region genes in the construct may be in non-murine vertebrate (for example, human) or murine (mouse) germline configuration. Non-murine vertebrate (for example, human) germline configuration will be suitable for achieving similar selection and maturation (recombination) events in the V, D and J regions to those found in the non-murine vertebrate (for example, human). Re-positioning of the human V, D and J region genes in the construct to mirror mouse germline configuration will allow efficient selection and maturation (recombination) events of the non-murine vertebrate (for example, human) V, D and J regions within the mouse.
The non-murine vertebrate (for example, human) V, D and J domains of the binding molecule in this aspect of the invention preferably result from recombination of the non-murine vertebrate (for example, human) V, D and J region genes. The process of somatic hypermutation will allow development of a diverse αH-chain-only antibody repertoire.
The nucleic acid construct in the L-chain deficient mouse used in the method may be integrated upstream of endogenous mouse αCH region genes. This will allow in vivo functional silencing of the endogenous αCH1 gene and formation of the H-chain-only IgA binding molecule. Alternatively, the nucleic acid construct in the mouse may comprise one or more mouse αCH region genes including an αCH1 gene. Here, in vivo functional silencing of the introduced construct αCH1 gene allows formation of the H-chain-only IgA binding molecule. In both cases, the αCH1 gene may be functionally silenced by class switch recombination.
The L-chain deficient mouse used in the method may have a nucleic acid construct integrated in the endogenous mouse genome, in which the nucleic acid construct comprises:
(i) non-murine vertebrate (for example, human) heavy chain V, D, J and C genes, for example in non-murine vertebrate (for example, human) or mouse germline configuration, including a non-murine vertebrate (for example, human) αCH1 gene; and
(ii) class switch recombination sequences upstream of the non-murine vertebrate (for example, human) αCH1 gene.
The class switch recombination sequences may facilitate class switch recombination-mediated functional silencing of the non-murine vertebrate (for example, human) αCH1 gene in vivo, thereby allowing production of a non-murine vertebrate (for example, human) H-chain-only IgA binding molecule in the mouse.
Class switch recombination sequences for use in the invention, for example mouse class switch recombination sequences, are as known in the art (see for example references 8 and 24, incorporated herein by reference in their entirety). These sequences facilitate the class switch recombination process to allow functional silencing of the αCH1 gene by deletion.
The L-chain deficient mouse used in the method may be inbred through two or more generations to increase production of the H-chain-only IgA binding molecules.
The L-chain deficient mouse used in the method preferably comprises a functional C, gene segments to allow pre-BCR B cell development and/or surface IgM expression.
In another aspect of the invention, there is provided an H-chain-only IgA binding molecule obtainable according to the method as described herein, or a functional fragment or derivative thereof.
The H-chain-only IgA binding molecule (including a functional fragment or derivative thereof) may have features as described above and below.
The H-chain-only IgA binding molecule may be in an isolated and/or substantially pure form. The binding molecule may be isolated and/or characterised using methods well known in the art. Once characterised, the binding molecule may be manufactured using recombinant or synthetic methods, also well known in the art.
The H-chain-only IgA binding molecule may be modified to increase solubility, for example by genetic engineering of one or more genes encoding the H-chain-only IgA binding molecule.
The H-chain-only IgA binding molecule of the invention may be specific to an antigen. The binding molecule may be engineered to be a bi- or multi-valent binding molecule with one or more specificities.
The H-chain-only IgA binding molecule of the invention may be monoclonal.
The H-chain-only IgA binding molecule may be non-human or part-human.
The H-chain-only IgA binding molecule may be obtained from mouse serum or secreted fluid (for example, milk, saliva, tears and/or sweat). Alternatively, the molecule may be obtained from the mouse faeces and/or urine.
The H-chain-only IgA binding molecule of the invention in certain embodiments does not include one or more or all camelid VHH-specific mutations found in VH to VHH substitutions in framework 2 (i.e. Val37Phe, Gly44Glu, Leu45Arg and Trp47Gly). Such mutations are not required for production of the binding molecule in L-chain-deficient mice.
The H-chain-only IgA binding molecule of the invention in certain embodiments does not include extended CDR3 region found in camelid H-chain-only antibodies (97, 40, 41, 42).
The invention encompasses a human H-chain-only IgA molecule obtainable according to methods described herein, other than known human H-chain-only IgA mutant proteins associated with αHCD (as described in references 84, 85, 43, which are incorporated herein by reference in their entirety).
The H-chain-only IgA binding molecule of the invention may be used as a screening agent, a diagnostic agent, a prognostic agent or a therapeutic imaging agent. The binding molecule may additionally or alternatively be used as an intracellular binding agent, or an abzyme. Accordingly, use of H-chain-only IgA binding molecule of the invention as a screening agent, a diagnostic agent, a prognostic agent, a therapeutic imaging agent, an intracellular binding agent or an abzyme is also within the scope of the invention.
Another aspect of the invention provides an H-chain-only IgA binding molecule as defined herein for use as a medicament.
Also provided is an H-chain-only IgA binding molecule as defined herein for use in the manufacture of a medicament for the treatment of a disease.
Further provided is an H-chain-only IgA binding molecule as defined herein for use in the discovery of a medicament. Use of the binding molecule in the discovery of a medicament is also encompassed.
Also provided according to the invention is an H-chain-only IgA binding molecule obtainable according to the invention method and modified, improved and/or evolved using an in vitro display system.
The invention further provides an H-chain-only IgA binding molecule-producing cell obtainable from an L-chain deficient mouse as defined herein. The cell may for example be a bone marrow cell, a mucosa cell, or a spleen lymphocyte cell. The cell may be a spleen lymphocyte IgA+ B220+ cell. As elaborated in the specific embodiments below, we have found that such a cell exhibits a novel B cell receptor.
According to another aspect of the invention there is provided a method for isolating an antibody-producing cell which produces an antigen-specific H-chain-only IgA binding molecule, comprising the steps of:
(i) obtaining an L-chain deficient mouse with functionally silenced endogenous lambda and kappa L-chain loci;
(ii) immunising the mouse with an antigen;
(iii) selecting for a cell producing an antigen-specific H-chain-only IgA binding molecule lacking a functional αCH1 domain following in vivo functional silencing of a gene encoding the αCH1 domain; and
(iv) isolating the cell selected in step (iii).
In this aspect of the invention, the L-chain deficient mouse may be produced using the method described in WO03/000737.
Selection and isolation of the cell may employ flow-cytometry, for example for the identification and isolation of B220+, syndecan+ spleen-derived cells in which an antigen-specific H-chain-only IgA binding molecule lacking a functional αCH1 domain is produced.
Another embodiment of this aspect of the invention is a method for isolating an antibody-producing cell which produces an antigen-specific H-chain-only IgA binding molecule, comprising the steps of:
(i) obtaining an L-chain deficient mouse with functionally silenced endogenous lambda and kappa L-chain loci using the method described in WO03/000737;
(ii) immunising the mouse with an antigen;
(iii) isolating sub-populations of cells from spleen, bone marrow and/or mucosa in the mouse;
(iv) screening the sub-populations of cells isolated in step (iii) by RT-PCT using JH to αCH2 amplifications to detect mutant α H chain transcripts in which the αCH1 exon has been deleted; and
(v) selecting those sub-populations of cells screened in step (iv) which have mutant a H chain transcripts; and
(vi) isolated a cell selected in step (v).
Further provided is an H-chain-only IgA binding molecule-producing hybridoma obtainable by fusion of a B-cell tumor line cell with the H-chain-only IgA binding molecule-producing cell as defined herein. Well known methods of generating and selecting single clone hybridomas for the production of monoclonal antibodies may be adapted for use in the present invention.
In another aspect of the invention, there is provided a medicament comprising an H-chain-only IgA binding molecule or functional fragment thereof as defined herein. The medicament may be formulated using well-known methods prior to administration into a patient.
The medicament may for example be formulated with a pharmaceutically or therapeutically acceptable excipient or carrier. Such excipients or carriers include a solid or liquid filler, diluent or encapsulating substance which does not interfere with the effectiveness or the biological activity of the H-chain-only binding molecule and which is not toxic to the host, which may be either humans or animals, to which it is administered. Depending upon the particular route of administration, a variety of pharmaceutically acceptable carriers such as those well known in the art may be used. Non-limiting examples include sugars, starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline, and pyrogen-free water.
Also provided is a method of treating a disease, comprising the step of administering a medicament as defined above to a patient in need of same.
The medicament may be administered by the route selected from the group consisting of orally, intramuscularly, intravenously, intradermally, cutaneously, topically, locally, ocularly and inhalation. Other suitable modes of administration are also contemplated according to the invention. For example, administration of the medicament may be via subcutaneous, direct intravenous, slow intravenous infusion, continuous intravenous infusion, intravenous or epidural patient controlled analgesia (PCA and PCEA), intrathecal, epidural, intracistemal, intraperitoneal, transdermal, transmucosal, buccal, sublingual, transmucosal, intranasal, intra-atricular, intranasal or rectal routes. The medicament may be formulated in discrete dosage units and can be prepared by any of the methods well known in the art of pharmacy.
All suitable pharmaceutical dosage forms are contemplated. Administration of the medicament may for example be in the form of oral solutions and suspensions, tablets, capsules, lozenges, effervescent tablets, transmucosal films, suppositories, buccal products, oral mucoretentive products, topical creams, ointments, gels, films and patches, transdermal patches, abuse deterrent and abuse resistant formulations, sprays, sterile solutions suspensions and depots for parenteral use, and the like, administered as immediate release, sustained release, delayed release, controlled release, extended release and the like.
Further provided according to the invention is an isolated nucleic acid (for example, a vector such as an expression vector, a BAC, a YAC or an artificial chromosome) encoding an H-chain-only IgA binding molecule as defined herein. The nucleic acid may comprise or contain any novel sequence disclosed herein (see for example in Tables 5 and 6 and/or
Sequence identity between nucleotide sequences can be determined by comparing an alignment of the sequences. When an equivalent position in the compared sequences is occupied by the same base, then the molecules are identical at that position. Scoring an alignment as a percentage of identity is a function of the number of identical amino acids or bases at positions shared by the compared sequences. When comparing sequences, optimal alignments may require gaps to be introduced into one or more of the sequences to take into consideration possible insertions and deletions in the sequences. Sequence comparison methods may employ gap penalties so that, for the same number of identical molecules in sequences being compared, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. Calculation of maximum percent identity involves the production of an optimal alignment, taking into consideration gap penalties.
Suitable computer programs for carrying out sequence comparisons are widely available in the commercial and public sector. Examples include the MatGat program (Campanella et al., 2003, BMC Bioinformatics 4: 29), the Gap program (Needleman & Wunsch, 1970, J. Mol. Biol. 48: 443-453) and the FASTA program (Altschul et al., 1990, J. Mol. Biol. 215: 403-410). MatGAT v2.03 is freely available from the site “http://bitincka.com/ledion/matgat/” and has also been submitted for public distribution to the Indiana University Biology Archive (IUBIO Archive). Gap and FASTA are available as part of the Accelrys GCG Package Version 11.1 (Accelrys, Cambridge, UK), formerly known as the GCG Wisconsin Package. The FASTA program can alternatively be accessed publically from the European Bioinformatics Institute (http://www.ebi.ac.uk/fasta) and the University of Virginia (http://fasta.biotech.virginia.edu/fasta_www/cgi). FASTA may be used to search a sequence database with a given sequence or to compare two given sequences (see http://fasta.bioch.virginia.edu/fasta_www/cgi/search_frm2.cgi). Typically, default parameters set by the computer programs should be used when comparing sequences. The default parameters may change depending on the type and length of sequences being compared. A sequence comparison using the MatGAT program may use default parameters of Scoring Matrix=Blosum50, First Gap=16, Extending Gap=4 for DNA, and Scoring Matrix=Blosum50, First Gap=12, Extending Gap=2 for protein. A comparison using the FASTA program may use default parameters of Ktup=2, Scoring matrix=Blosum50, gap=−10 and ext=−2.
A polypeptide encoded by the nucleic acid as defined herein is also encompassed by the invention.
In another aspect there is provided a host cell comprising the nucleic acid of the invention.
As used herein, the term “binding molecule” refers to an antibody produced in vivo by an animal such as mouse, generated for example in response to an antigen, and also where appropriate to a synthetic binding molecule which mimics the binding ability of an antibody, for example with modified binding or other pharmacological properties. Where appropriate, the term also encompasses functional binding molecule fragments, for example antigen-binding or effector antibody fragments, and/or functional derivates thereof.
As used herein, the term “in vivo” refers to an endogenous or a natural (non-engineered) process. The endogenous or natural process occurs spontaneously.
The term “in vivo functional silencing of a gene encoding the CH1 domain” includes deletion of all or part of the gene encoding the CH1 domain, such that no functional protein can be expressed from the domain. Suitably, “in vivo functional silencing of a gene encoding the CH1 domain” occurs spontaneously by class switch recombination.
Functional silencing of the light chain loci may be achieved by disruption (e.g., by insertion into the locus), or deletion of all or part of the loci, such that no functional protein can be expressed from the loci.
The term “mouse” used herein encompasses in further aspects of the invention other vertebrates such as mammals, preferably non-human mammals such as other rodents including rats.
As used herein, a non-murine vertebrate includes mammals such as a rat and a human, particularly a human.
Somatic alterations leading to CH1 deletion occur at low frequency. This is a limiting step in H-chain-only IgG production, however L-chain deficient mice homozygous in the germline for deletion or disruption of a CH1 exon, such as a γ CH1 exon or alpha CH1 exon, allow H-chain-only antibodies, such as H-chain-only monoclonal antibodies, with defined specificities to be produced.
Accordingly, in an alternative embodiment, the present invention also provides an L-chain deficient mouse having functionally silenced endogenous lambda and kappa L-chain loci and lacking a functional CH1 domain in the germline. Preferably the lambda and kappa L-chain loci are silenced by disruption, such as by an insertion, or by deletion of all or part of the locus such that light chains cannot be expressed from the loci. Preferably the CH1 domain is disrupted (e.g., by insertion), or fully or partially deleted from the germline such that the CH1 domain can not be expressed. Preferably the CH1 domain is a gamma or alpha CH1 domain. Preferably the mouse is homozygous for deletion and/or disruption of the lambda light chain locus, kappa light chain locus and CH1 domain.
The mouse having functionally silenced endogenous lambda and kappa L-chain loci may, for example, be made as disclosed in WO03/000737, which is hereby incorporated by reference in its entirety. In WO03/000737 functional silencing of the Igκ locus was achieved by insertion of neo into Cκ (46); functional silencing of the λ locus was by a Cre-IoxP mediated deletion of ˜120 kb encompassing all Cλ genes.
The mouse may also have a functionally silenced endogenous heavy chain locus, for example produced as disclosed in WO04/076618, which is hereby incorporated by reference in its entirety. In WO04/076618 functional silencing of the endogenous heavy chain constant region locus was achieved by Cre-IoxP mediated deletion of the heavy chain constant region genes.
Preferably the mouse is capable of expressing preBCR and/or surface display of an endogenous or exogenous IgM.
The mouse of the invention in this aspect, lacking a functional CH1 gene in the germline, may additionally comprise a nucleic acid construct integrated into the endogenous mouse genome, in which the nucleic acid construct comprises non-murine heavy chain genes from which the H-chain-only antibody is produced.
The non-murine heavy chain genes may be from other vertebrates including mammals such as from a rodent, like rat or rabbit, or particularly from a human.
One example of a suitable construct comprising human heavy chain genes is the IgH YAC construct disclosed in WO2004/049794 and/or reference 80, which are hereby incorporated by reference in their entirety.
According to this aspect of the invention there is provided a transgenic L-chain deficient mouse having functionally silenced endogenous lambda and kappa L-chain loci and lacking a functional CH1 domain in the germline, preferably lacking a functional αCH1 domain or γCH1 domain, and having a nucleic acid construct integrated in the endogenous mouse genome, in which the nucleic acid construct comprises non-murine vertebrate (for example, human) V, D and J region genes and in which the mouse produces a mouse-non-murine vertebrate (such as mouse-human) chimeric H-chain-only antibody comprising non-murine vertebrate (for example, human) V, D and J domains and one or more mouse CH domains excluding a functional CH1 domain.
The invention further provides a method for producing a H-chain-only immunoglobulin, preferably a H-chain-only immunoglobulin G or A, in an L-chain deficient mouse having functionally silenced endogenous lambda and kappa L-chain loci and lacking a functional CH1 domain, preferably lacking a functional gamma CH1 domain or lacking a functional alpha CH1 domain, comprising the steps of:
The method may comprise the further step (iii) of isolating the H-chain only antibody.
The H-chain only antibody may be produced in response to antigen challenge, such as immunisation with a specific antigen.
In an aspect there is provided a cell from a L-chain deficient mouse having functionally-silenced endogenous lambda and kappa L-chain loci and lacking a functional CH1 domain, said cell being capable of expressing a H-chain-only immunogloblulin, preferably the CH1 domain is a gamma or alpha CH1 domain and the H-chain-only immunogloblulin is IgG or IgA respectively.
In an aspect there is provided a hybridoma cell obtainable by fusion of a B-cell tumor line cell with a cell from a L-chain deficient mouse having functionally silenced endogenous lambda and kappa L-chain loci and lacking a functional CH1 domain, said cell being capable of expressing a H-chain-only immunogloblulin, preferably the H-chain-only immunogloblulin is IgG or IgA, i.e. the CH1 domain is a gamma CH1 domain or an alpha CH1 domain.
Particular non-limiting embodiments of the present invention will now be described below with reference to the following drawings, in which:
indicated in NM) are present in lymphoid tissue, sometimes accompanied by the full size product (˜650 bp). β-actin served as a reference (25 cycles). (B) RT-PCR amplification of bone marrow (bm) and spleen (sp) from normal and L−/− mice using a J/hinge oligo, specific for J to hinge joins that lack CH1 (JH1, JH2 or JH4 and γ2a or γ2b), in combination with the γCH2c oligo. In comparative control reactions JH2 to γCH2a and β-actin (21 cycles) was amplified. (C) For the analysis of spleen cells by FACS and RT-PCR from L−/− mice the lymphocyte gate, established by forward (FS) and side scatter (SS), was set to include large cells (P1). These cells were collected (P2-P4) according to their staining profiles for B220. Large B220+ cells (P3) show a γ H-chain RT-PCR band, from JH4 to γCH2, of reduced size (˜350 bp) lacking CH1 whilst other cell fractions contain a normal size H-chain transcript (˜650 bp). PCR reactions were normalized using β-actin. (D) Surface staining for B220 and cytoplasmic staining for IgG showed in confocal images that H-chain antibody producing B-cells are of larger size (D2). DIC denotes Differential Interference Contrast and the size bars are 10 μm. (E) Surface staining for syndecan (syn) (CD138) identified a population (S3) only expressing H-chain transcripts without CH1 in L−/− mice. Syn+ cells from NM mice, which are lacking in CΔ mice, established the gate for the cell sort. Normalized RT-PCR reactions (32 cycles) were carried out with the sensitivity and specificity being verified by increased levels of unsorted spleen cells (10×, 100×). Control reactions without DNA (−) are indicated. The data are representations using different mice in at least 3 independent experiments giving very similar results;
In the following example we report that the absence of L-chain does not prevent serum antibody production in mice. Quite unexpectedly, we found antibodies in the serum of L-chain deficient mice without any further genetic manipulation other than functional silencing of the lambda and kappa L-chain loci. Diverse H-chain-only IgG without CH1 is secreted despite compromised B cell development. We show that H-chain-only IgGs are produced from transcripts lacking the CH1 exon, and we identify in some somatic cells different genomic deletions that can give rise to these transcripts. The results show that L-chain deficient animals are a useful tool for the production of H-chain-only antibodies such as therapeutic H-chain-only antibodies.
Mice. The derivation of Igκ and Igλ deficient (L−/−, with Cκ disrupted by neo insertion and a ˜120 kb region from Cλ2 to Cλ1 removed by targeted integration and Cre-IoxP deletion), Cμ truncation (μNR, lacking Cμ1 and Cμ2 by targeted integration of neo), C deletion (CΔ, lacking a ˜200 kb region from Cμ to 3′ of Cα removed by targeted integration and Cre-IoxP deletion) and μMT mice has been described (7, 17, 45, 6). L−/− and μNR animals were crossbred to homozygosity. Mice, 12-28 weeks old, were analyzed and compared with littermates or age-matched controls.
ELISA and immunization. Serum antibodies were analyzed as described (46) on Falcon plates coated with 10 μg/ml anti-mouse IgM, Igκ (Sigma) or IgG (Binding Site). Biotinylated detection antibodies were anti-mouse IgM (Sigma), IgG (Amersham), Igκ (Zymed) and Igλ (BD Pharmingen). To determine the antibody concentration purified IgG (DB3) was used (47). Immunisations were carried out with 100 μg OVA in CFA (s.c.) and subsequently 50 μg OVA in FA (i.p.), 30 and 14 days later, respectively. Ig secretion was identified by ELISA on plates coated with 10 μg/ml OVA (Sigma).
Western analysis. Serum Ig was incubated with anti-mouse Ig(μ, γ and α H-chain specific)-coupled Sepharose, separated on Ready-Gels (Bio-Rad) and transferred to nitrocellulose membranes as described (27). Filters were incubated with biotinylated anti-mouse Ig: μ-chain (Sigma), γ-chain (Amersham) and κ (Zymed) and λ (BD Pharmingen) L-chain specific. This was followed by incubation with streptavidin biotinylated HRP solution (Amersham) and visualization of bands using SuperSignal West Pico chemiluminescent substrate (Pierce, Illinois). Protein mol wt standards were supplied by Bio-Rad and Fermentas.
Flow cytometry. Bone marrow, spleen and peritoneal cell suspensions were prepared and multi-color analyses were carried out on a BD FACSCalibur. Cells were stained, in combination, with labeled anti-mouse Ig recognizing CD45R (B220) either PE- or APC- or BIO-conjugated, PE-conjugated anti-c-kit (CD117), BIO-conjugated anti-CD43, BIO-conjugated anti-CD25, FITC-conjugated anti-IgD, PE-conjugated anti-Igκ, FITC-conjugated anti-Igλ, PE-conjugated anti-CD5 (Ly-1), FITC-conjugated anti-CD79b (Igβ), FITC-conjugated anti-mouse CD21/35 (all from BD Pharmingen) and FITC-conjugated anti-IgM (Zymed). Reactions with BIO-conjugated antibodies were subsequently incubated with Tri-color-conjugated streptavidin (Caltag). CellQuest software (BD Biosciences) was used for the analysis.
For sorting on a BD FACSAria, spleen cells were stained with PE-conjugated anti-CD45R and, separately, FITC-conjugated anti-CD45R and BIO-conjugated anti-CD138 (syndecan-1) (BD Pharmingen) followed by incubation with PE-Cy5.5-conjugated streptavidin (eBioscience). For the analysis of cytoplasmic Ig, spleen cells stained for surface CD45R, were incubated with Fc-specific FITC-conjugated anti-IgG (Sigma) using a fix and perm cell permeabilization kit (Caltag). IgG positive cells were collected and viewed on a Zeiss confocal microscope (LSM 510 META) and images were obtained using Zeiss LSM 3.2 software.
RT-PCR analysis. RNA was isolated from tissue or sorted cells using Trizol (Gibco-BRL) and reverse-transcribed at 42° C. with Omniscript reverse transcriptase (Qiagen). PCR reactions with JH and γCH2a primers were set up using KOD Hot Start DNA polymerase (Novagen), at a final MgSO4 concentration of 0.8 mM. The cycling conditions were 94° C. for 2 min, 40 cycles of 94° C. for 15 sec, 58° C. for 30 sec, 72° C. for 15 sec, followed by 72° C. for 10 min. Amplification with VH specific primers was as above, but with a 52° C. annealing temperature for Vgeneric, V3609 and VS107/J606. RT-PCR products were either sequenced directly or cloned by adding a 3′ A overhang and using a TA Cloning Kit (Invitrogen). β-actin PCR was performed as above but with an annealing temperature of 61° C. The J/hinge to γCH2c PCR was set up as above but with a touchdown program: 94° C. for 2 min, 21 cycles of 94° C. for 15 sec, 69° C. (−0.33° C./cycle) for 30 sec, 72° C. for 10 sec, followed by 25 cycles of 94° C. for 15 sec, 62° C. for 30 sec, 72° C. for 10 sec. For linear amplification of cDNA ends first-strand cDNA synthesis and primer extension was performed on 1 μg of total RNA as described (BD SMART mRNA Amplification Kit). Double stranded cDNA was purified using Wizard SV Gel PCR Clean-Up System (Promega) and linear amplification of the 5′ ends was carried out using KOD and nested γCH2 primers (γCH2b and γCH2a) as described (48). The product from the fourth round of amplification was separated by agarose gel electrophoresis and bands between 700 and 1200 by were excised and purified using the Wizard kit. The purified fragments were cloned as above and sequenced. All primer sequences used are listed in Table 1 below. Agarose gels were run with size markers in kb (Hyperladder 1, Bioline) and/or by (100 by DNA ladder, Invitrogen).
Genomic DNA analysis. Genomic DNA was prepared as described (27) with the addition of linear acrylamide to aid the precipitation of small amounts of DNA from the sorted cells. Long PCR was carried out with Platinum PCR Supermix High Fidelity (Invitrogen). Reactions were set up with DNA from 1-2×103 sorted cells (˜10 ng), equivalent amounts of ES cell or hybridoma DNA and 100 nM of each primer. The reactions with unsorted spleen DNA contained ˜100 ng DNA. An initial denaturating step of 94° C. for 1 min was followed by 94° C. for 15 sec and 68° C. for 15 min. A first round PCR of 20-36 cycles from VDJ or JH4long to γCH2e was followed by a nested second round PCR of 15-30 cycles from 3′ Eμ to γCH2d, δ2aCH2long or γ2bhingelong. Any bands obtained were cloned as described above and sequenced.
Mass-spectrometry. Coomassie-stained bands were destained, reduced, carbamidomethylated and digested overnight with 10 ng/μl Sequencing Grade Modified Trypsin (Promega) in 25 mM NH4HCO3 at 30° C. The resulting peptide mixtures were separated by reversed-phase liquid chromatography on a Vydac C18 column (0.1×100 mm, 5 μm particle size), with a gradient of 0-30% acetonitrile over 30 min, containing 0.1% formic acid, at a flow rate of 500 nL/min. The column was coupled to a nanospray ion source (Protana Engineering) fitted to a quadrupole-TOF mass spectrometer (Qstar Pulsar i; Applied Biosystems/MDS Sciex). The instrument was operated in information dependent acquisition mode, with an acquisition cycle consisting of a 0.5 sec TOF scan over the m/z range 350-1500 followed by 2×2 sec MS/MS scans (triggered by 2+ or 3+ ions), recorded over the m/z range 100-1700. Proteins were identified by database searching of the mass spectral data using Mascot software (Matrix Science).
RNA-FISH. Sorted cells were fixed on slides and analyzed by two-color RNA FISH. Methods for the analysis, with probes for FISH generated previously, have been described (ref 23 and refs therein). Images were visualized using Olympus BX40 and BX41 microscopes. Experiments were performed 2 to 4 times and at least 100 nuclei or 200 alleles were counted each time.
IgG Expression without L-Chain
We initially aimed to investigate whether mechanisms for single H-chain Ig expression are naturally present in the mouse and are used if production of conventional antibodies is prevented. This was examined by using mice with silenced Igκ and Igλ L-chain loci (L−/−) obtained by gene targeting (7). In the L−/− mice all CL genes are either disrupted (Cκ, Cλ1) or removed (Cλ2, Cλ4 and Cλ3) which prevents the production of functional L-chain (
To establish unambiguously whether surface IgM expression is essential to drive H-chain IgG expression we crossed L−/− mice with μMT animals, which carry a targeted disruption of the μ transmembrane exons (6). Serum analysis of heterozygous and homozygous littermates established that H-chain-only IgG secretion is only operative when the transmembrane configuration of Cμ is unaltered (
Whereas B cell differentiation, resulting from the presence of truncated IgM did not increase the level of H-chain-only IgG produced by L−/− mice the amounts could be considerably increased by a conventional immunization regime (
To determine the mol wt and assembly of these novel murine H-chain-only antibodies, Western blot analysis was performed on serum Ig separated under reducing and non-reducing conditions (
Identification of substantial amounts of diverse H-chain-only Ig in the serum of mice lacking L-chains prompted extensive analysis of B cell differentiation events using flow cytometry (
The cells in μNRL−/− mice that have acquired the expression of a H-chain-only BCR may overcome the block in conventional B cell differentiation and be released into the periphery as mature B cells. Proliferation of such cells may explain the distinct B220+CD21/35+ B cell population of splenic lymphocytes in L−/− mice (
H-chain transcripts lacking CH1 are generated in L−/− and normal mice The production of γ H-chain transcripts in different tissues from L−/− mice was assessed by RT-PCR using JH to γCH2 amplifications, which in normal animals produces a ˜650 by band in lymphoid tissue (
In some JH to γCH2 amplifications the normal mouse spleen cDNA control gave a faint ˜350 by band ( indicated) which on sequencing was found to lack CH1 (data not shown). This smaller band is frequently obscured due to amplification of an abundance of normal size products. However, the presence of this band implies that normal mice can generate transcripts, which could produce H-chain-only antibodies. To investigate whether H-chain transcripts lacking γCH1 are regularly produced in normal mice we designed oligonucleotides that would only recognize splice products where a JH segment joins a γ2a or γ2b hinge exon. Surprisingly, in normal mice ˜340 by JH-hinge transcripts are readily found in the spleen and frequently, but not always, in bone marrow (
aNumbers refer to the full sequences in FIG. 7 and Table 4 with 028 to 129 from unsorted spleen cells and 132 to 213 from syn+ spleen cells.
CCTAC
aMutational differences not found in corresponding germline gene segments from 129, BALB/c or C57BL/6 mouse strains are underlined.
bFor clone details see Table 2.
cCorresponding genomic sequence identified.
Flow cytometry using established separation parameters (e.g. scatter gating) did not reveal any obvious candidates or distinct B cell populations that showed enrichment for the truncated transcripts. So the lymphocyte gate was extended to include larger or differently shaped cells in the sorting process (20, 21) as antibody production and conceivably H-chain Ig secretion could be linked to an increase in cell size (
To understand whether IgG+B cells from L−/− mice bear the features of conventional antibody secretors and thus are the product of normal lymphocyte differentiation events we carried out stainings for syndecan (CD138), which identifies plasma cells, in combination with B220 (22). Syndecan+ cells (S3) in
Encouraged by the diversity of the H-chain antibodies found, we investigated whether activation of the IgH locus and diversity of VH gene usage is equally operative in L−/− mice compared to normal animals (23, 24). To detect individual IgH alleles we used RNA-FISH with a probe, Iμ, which establishes locus activity (
RT-PCR and sequence analysis of smaller size H-chain bands identified a lack of γCH1 or, in a few cases, both the γCH1 and γhinge exons (Table 3). One way to identify mutations leading to H-chain-only antibodies is the derivation of hybridomas. Another approach of gaining access to cells expressing IgG is by sorting for syndecan positive cells. In order to enrich this starting material with DNA from cells expressing IgG, we set up an assay for amplifying switched γ regions. A long range PCR with a JH primer and a γCH2 primer, followed by a second nested reaction from 3′Eμ to γCH2d (
To establish unambiguously whether transcripts that lack CH1 are the result of genomic deletions we derived forward primers from their D-JH junction sequence. Successful amplification and cloning from a rearranged VH of the 7183 family identified a large deletion removing the μ/γ switch region and CH1 of Cγ2b concluding 107 nucleotides 5′ of the hinge exon (clone 029, Tables 3 and 4,
In L-chain deficient mice, B cell development is arrested at the pre B2 to immature B cell stage in the bone marrow (7). At this transition stage, IgM, comprising a μ H-chain covalently linked with a κ or λ L-chain in dimeric configuration, should be expressed on the cell surface associated with the co-receptor chains Igα/β. Developmental progression of a compromised BCR lacking any of these chains is normally blocked (6-8, 26), so the finding of IgG in the serum of L-chain deficient mice came as a surprise. Analysis by Western blot and mass spectrometry indicated that these proteins were lacking the CH1 domain. This was confirmed by RT-PCR, which identified short γ transcripts lacking CH1 (or in some instances CH1 and hinge) in the lymphoid organs of L-chain deficient mice. These findings are in agreement with reports for transgenic mice that express H-chain only Ig, where the loss of CH1 appears to be essential (27-29). The shorter nascent-translated H-chain cannot form a complex with the H-chain binding protein as it lacks the association sites in CH1 (15, 30). This would result in unhindered transport through the ER allowing surface deposition, as well as H-chain secretion. The stability of such H-chain-only Ig is remarkable and it can be argued that the lack of CH1 and the loss of chaperone association may prevent degradation of a basically incomplete Ig. Sorting experiments indicated that the main sources of short transcripts were large, syndecan positive cells, i.e. plasma cells, and thus the product of normal lymphocyte differentiation, while conventional transcripts were abundant in a B220high cell population. However, these results raised the question of how the protein deletions occurred and how antibody-producing cells could be generated in the absence of noticeable BCR expressing B cells.
Different mechanisms can lead to exon removal such as alternative splicing, splice-site mutations or exon deletions; all of which have been found for Ig genes (31, 32). If expression of H-chain IgG were controlled at the transcriptional stage, for example by selection of splice products leading to polypeptides which could be released from the cell, then the rearranged H-chain gene should be unaltered. To look for the existence of somatic mutations, we sorted syndecan positive cells, which are enriched for cells producing transcripts lacking CH1, and extracted the DNA. Long range DNA-PCR analyses using 5′ primers in the JH and Eμ region and reverse primers in the γCH2 exon ensured that only switched γ genes were amplified. With this approach, we were able to clone three different genomic Cγ deletions where most or all of CH1 and the μ/γ switch-region were removed, but the hinge exon and Eμ intron enhancer downstream of JH, were left intact. With these modifications transcription levels and splicing from a rearranged VHDJH to the hinge exon should be maintained, producing a truncated protein. A putative mechanism for CH1 deletion suggested by our sequence data is error during the class-switch process. The switch-region upstream of each CH gene is highly repetitive, several kb in length and accommodates repairs to DNA lesions, such as double strand breaks. The recombination itself, which removes Cμ and juxtaposes the rearranged VHDJH close to a downstream CH gene, occurs between non-homologous sequences without any consensus motif defining precisely the donor and acceptor breakpoints (33). It is possible that imprecise switching removes all or part of CH1, which would allow Ig surface expression.
However, switching (and presumably faulty switching) occurs from mature IgM-expressing lymphocytes, which are difficult to identify in the spleen of L−/− mice, occurring as a small population of B cells expressing high levels of B220. It is possible that failing to become a mature IgM-expressing B cell initiates early class-switching which may explain why serum IgM is absent in L−/− mice and camelids do not appear to produce H-chain-only IgM or VHHDJH-Cμ transcripts (34, 35). This possibility is strengthened by the fact that we can identify in the spleen full-length y transcripts that are much more abundant and diverse than short transcripts. Presumably, the switch from μ to γ occurs in a large number of cells, but in most cases, this does not allow production of a H-chain that can be transported to the cell surface without L-chain. Only when faulty switching gives rise to DNA sequences encoding transcripts lacking CH1 would the B cell be selected for survival. The absence of H-chain IgG in L−/−μMT−/− mice suggests that a pre-BCR dependent proliferative stage is required; probably to produce the number of cells required to obtain these specific aberrant switching events. A knock-in gene encoding a mutant μ chain (μNR, ref 17) was introduced into the genome of L−/− mice, which resulted in expression of a truncated μ H-chain on the cell surface in the absence of L-chain. This ensured cell survival but did not result in an increased IgG level, which could be seen as puzzling, as μNR mice expressing L-chain have a normal, high level of IgG. However, the pre-BCR dependent proliferative stage is slightly impaired in the μNR mice. Also, in μNR and μNRL−/− mice, the expression of truncated μ significantly increases the number of a particular CD5+ lymphocyte subset, (CD5+ B1a cells), which rarely switch (36, 37). In addition to the deletion of the CH1 region, we have identified CH1 point mutations in some of the switched γ regions from L-chain deficient mice. However, none of them corresponds to a typical splice site mutation so it is not clear if these changes affect splicing. Further analysis is required to determine whether they cause exon skipping by altering exon recognition by cis-elements involved in the splicing process (38). If this is the case L-chain deficient mice might provide useful information on the mechanism controlling splice site usage.
Evidence that secretion of H-chain-only Ig in L-chain deficient mice is antigen-dependent, comes from the increased titers of specific antibody after immunization. Also several functional VH sequences were found, which harbored mutations that can be attributed to somatic hypermutation (39). We investigated whether specific alterations compensate for the lack of L-chain association, as found in adapted camelid VHH exons (34). From the alignment of VHH sequences with the V regions of mouse H-chain antibodies it was found that this was not the case. None of the hallmark amino acids found in VH to VHH substitutions in framework 2 (Val37Phe, Gly44Glu, Leu45Arg and Trp47Gly) (40, 41) were seen, and in one case the reverse was found with an Arg to Leu change at position 45 (Kabat numbering). Some camelid H-chain Igs bear a long CDR3 and it has been suggested that CDR3s encompassing a more extensive D segment and/or substantial N-sequence additions may be an advantage to compensate for the smaller antigen-binding area of H-chain Ig compared to the conventional H-L Ig (34, 35, 40, 41). A longer CDR3 was not found with the mouse H-chain antibodies, but it should be noted that antigen-specific dromedary H-chain antibodies with shorter CDR3 (7aa) have also been identified (42).
Expression of H-chain-only IgG in L−/− mice appears to differ from human HCD. HCD are monoclonal B cell lymphoproliferations secreting mutant H-chain not associated with L-chain. It has been hypothesized that these proliferations are caused by expansion of cells that express altered H-chains because they have previously lost the ability to produce L-chains (although there are cases where free L-chains are produced by tumor cells) (43). The results we have obtained show that the absence of L-chains leads to selection of cells producing mutant H-chain lacking CH1, when normal competing B cells are absent. However, unlike in HCD, it appears from the Western blot analysis and the sequence data from L′−/− mice that gross alterations in the VH regions are not present in the majority of the cells. In addition, in L-chain deficient mice we have not observed the lymphoproliferations which occur in HCD.
The modifications observed in L-chain deficient mice produce a domain configuration comparable to that which has been identified in both camelids and cartilaginous fish; representing in the former a relatively recent adaptation (13) and in the latter a possible remnant of the primordial antibody structure that preceded the heterodimeric association of H- and L-chains (11). In lower vertebrates H-chain dimers have been recognized that lack a classical CH1 domain, important to provide the cysteine residue that forms the disulphide linkage with the L-chain (11, 12). The evolution of Ig domains, their multiplication and diversification to permit functional interaction, vividly illustrates the ongoing selective pressure on antibody genes. Specific alterations, in the case of the L−/− mice, the removal of CH1, prevented Bip association without affecting H-chain dimerisation, an essential requirement to secure the antibody structure for immune protection (44).
In conclusion, Example 1 shows that in mouse B cells the removal of CH1 permits cellular release of fully functional IgG antibodies without L-chain and development of a diverse H-chain-only antibody repertoire. Mouse VH genes can be expressed as H-chain antibodies without acquiring VHH specific changes and maintain their inherited sequence characteristics and lengths (41). A B cell repertoire with somatic hypermutation would be of great importance for the production of H-chain-only monoclonal antibodies in mice. Difficulties with generating hybridomas from L-chain deficient mice using whole organs (e.g. spleen) may be due to the small numbers of activated lymphoblasts present. This should be overcome for example by increasing the cell population of H-chain-only Ig producing progenitors. Since somatic alterations leading to CH1 deletion, due to its very low frequency, is a strong limiting step in H-chain-only IgG production, deleting a γ CH1 exon in the germline of L-chain deficient mice allows H-chain-only monoclonal antibodies with defined specificities to be readily produced.
In this example, we show that IgM lacking the BiP-binding domain is displayed on the cell surface and elicits a signal that allows developmental progression even without the presence of L-chain. The results are reminiscent of single chain Ig secretion in camelids where developmental processes leading to the generation of fully functional H-chain-only antibodies are not understood. Furthermore, in the mouse the largest secondary lymphoid organ, the spleen, is not required for H-chain-only Ig expression and the CD5 survival signal may be obsolete for cells expressing truncated IgM.
As noted above, classical antibodies consist of multiple units of paired H- and L-chains and are produced by all jawed vertebrates studied to date. In addition, Tylopoda or camelids (camels, dromedaries and llamas) secrete dimeric H-chain-only IgG (9) similar to single chain Ig also found in primitive cartilaginous fish (11, 12). Homodimeric H-chain-only antibodies in camelids, as well as in lower vertebrates, either lack the CH1 or a CH1-type domain, respectively, which normally provides the disulphide linkage with the L-chain (11, 12, 49). Both antibody-types are produced by DNA rearrangement of the IgH locus, where D (diversity), J (joining) and V region gene segments are recombined, initiating B cell development in B lymphocyte progenitor cells (reviewed in 4, 50). Juxtaposition of VH-D-JH is followed by expression of a μ H-chain, which associates with the surrogate L-chain consisting of VpreB and λ5 and the co-receptor polypeptides Igα and Igβ to form the preB cell receptor (preBCR). After expansion of the preB cell pool, a preBCR negative stage occurs where μ H-chain is intracellular and surrogate L-chain is not detected (51). Production of κ or λ L-chain upon VL to JL rearrangement allows surface IgM expression as part of the BCR; a requirement so that a sizable number of cells can colonize the secondary lymphoid organs, such as spleen or lymph nodes (7). Further differentiation to produce class-switched isotypes is performed by replacing Cμ with a 3′ CH gene, for example Cγ or Cα, in the class switch recombination process (52). The amino-terminal signal sequence of nascent H- and L-chains permits their shuttle to the lumen of the ER, which is accompanied by glycosylation and BCR assembly. Upon quality control of this reaction, disulphide linked chains are transported to the Golgi complex for further processing involving carbohydrate additions, which is followed by packaging into vesicles transported to the plasma membrane for release (53, 54). Analysis of IgM and IgG mutants have established that H-chain-binding protein (BiP, also termed GRP78) associates with the CH1 domain of newly-synthesized Ig H-chains preventing their cellular release unless BiP is replaced by L-chain (55). However, H-chains not associated with BiP can be exported with or without L-chain; thus the lack of CH1 in camelid H-chain IgG secures secretion. In humans the absence of VH or CH1 allows secretion without L-chain in rare B cell malignancies known as Heavy Chain Disease (HCD) (reviewed in 56). Also, in transgenic mice truncated H-chains can be released from the cell, not necessarily leading to malignant growth (17, 27, 57). Murine preB cells with full length μ H-chains on the cell surface without associated surrogate or conventional L-chain have also been described (58, 59, 60). More recently, the ability for some B cells to display H-chains on their surface in the absence of L-chains has been reported (61).
Our approach to understanding H-chain-only antibody expression and the importance of the Ig L-chain focused on the analysis of modified mice obtained by gene targeting and transgenesis. In Example 1 above we revealed that H-chain-only IgG can be produced naturally by removal of all or part of the CH1 exon of a Cγ gene. Here we show that expression of a L-chain deficient BCR occurs in a high number of cells when stable interaction of the H-chain with BiP is prevented. The occurrence of B-1a lymphocytes, in mice expressing truncated IgM without L-chain, is independent of CD5, the cell surface receptor characterizing this B cell subset. Aborted spleen development in Hox11 knock-out (spleenless) animals revealed that asplenia with its missing B cell lineages does not prohibit single-chain antibody secretion, indicative of B1 cell independent expression.
2.1 Materials and methods
Mice. The derivation of Cμ truncation (μNR), ΔVμ transgenic, Igκ and Igλ deficient (L−/−), CD5 deficient and Hox11 deficient mice has been described previously (7, 17, 62, 63, 64). Animals were crossbred to homozygosity to obtain the following new combination of features: μNR L−/−, μNR CD5−/−, μNR Hox11−/−, Hox11−/− L−/− and ΔVμ L−/−. Mice, 3-6 months old, were analyzed and compared with littermates or age-matched controls.
Flow Cytometry. Cell suspensions were prepared from bone marrow, spleen and peritoneal cells and multi-color analyses were carried out on a BD FACSCalibur. For the analysis cells were stained, in combination, with anti-mouse antibodies recognizing CD45R (B220) conjugated to either Phycoerythrin (PE), allophycocyanin (APC) or Biotin (BIO), PE-conjugated anti-c-kit (CD117), BIO-conjugated anti-CD43, BIO-conjugated anti-CD25, PE-conjugated anti-Igκ, FITC-conjugated anti-Igλ, PE-conjugated anti-CD5 (Ly-1), FITC-conjugated anti-CD79b (Igβ), FITC-conjugated anti-CD21/35 (all from BD Pharmingen), FITC-conjugated anti-IgM (Zymed) and FITC-conjugated anti-human IgM (Nordic). Reactions with BIO-conjugated antibodies were subsequently incubated with Tri-color-conjugated streptavidin (Caltag). CellQuest software (BD Biosciences) was used for the analysis.
ELISA. Serum antibodies were analyzed as described (Zou et al., 1995) on Falcon plates coated with 10 μg/ml anti-mouse IgM, Igκ (Sigma), Igλ (BD Pharmingen) or IgG (Binding Site) or anti-human IgM (Sigma). Biotinylated detection antibodies were anti-mouse IgM (Sigma), IgG (Amersham), Igκ (Zymed) and Igλ (BD Pharmingen) and anti-human IgM (Sigma). Standard deviation of the serum antibody titer was calculated from at least 5 age-matched mice except ΔVμ control where serum from 3 mice confirmed previous observations.
Antibodies without L-chain can be released from the cell as dimerised H-chains in H-chain-only Ig lacking CH1 or as monomers, dimers or polymers in HCD. In both malignant and healthy expression of H-chain-only Ig, no full-length polypeptides are produced (49, 65). Alterations, such as the removal of the first C region exon in Cμ or Cγ are responsible for permitting BiP(chaperone)-independent cellular transport and discharge of H-chains without initiating an unfolded protein response (UPR), which normally leads to degradation of incomplete polypeptides (21, 55). Mice that do not express antibody L-chains generate a wide range of differently rearranged IgG H-chains, which have many of the attributes of H-chain-only IgG found in camelids (see Example 1 above). We wanted to gain information as to how BiP-independent H-chain release from the cell affects developmental processes. Mice carrying different combinations of transgenic and knockout alterations affecting lymphocyte differentiation events were analyzed by comparing cell surface staining using flow cytometry.
The alterations to the Ig loci (μ, κ and λ), the lymphocyte cell-surface marker (CD5) and the homeobox gene responsible for spleen development (Hox11) are depicted in
Surface Assembly of IgM without BiP-Retention Domain and L-Chain
As shown in Example 1, spontaneous IgG expression without L-chain is possible in mice when the first Cγ exon is removed during switch-recombination. The role of Cμ and whether surface expression needs to be achieved at the transitional B cell stage before switching is unclear. For a μ H-chain to be expressed on the B cell surface without L-chain beyond the preB cell stage may require the BiP-retention domain to be removed. Analysis of B220+ IgM+ bone marrow cells from normal, μNR and μNR L−/− mice showed a reduction when CH1 and CH2 were missing, which was further reduced with the absence of the L-chain (
In both μNR and μNR L−/− mice, the levels of CD5+ B cells in the bone marrow are increased compared to normal mice, which agrees with previous observations that a shorter μ chain permits expansion of this particular B cell subset (17). However, this is not seen in bone marrow cells from μNR Hox11−/− compared to Hox11−/− mice (
The lack of the CD5 survival signal, which is important for negative BCR feedback signaling and stimulation of IL-10 production (67), might be expected to reduce the number of cells that express truncated IgM. Analysis of the bone marrow from μNR CD5−/− mice (
Developmental Progression from Primary to Secondary Lymphoid Organs
It was found that μNR chains were not only able to be expressed on the cell surface in the absence of L-chains, but were also efficient in supporting B cell development in the periphery. This is documented in the spleen by the occurrence of CD21/35+ cells, which are 11.9% in μNR L−/− or about ⅓ of what is found in μNR mice and ¼ of the numbers in normal mice (
Previous studies of peritoneal lymphocytes showed that the level of CD5+ B-1a cells is increased in μNR mice and that their lack in L−/− mice is accompanied by the appearance of larger or differently shaped cells (17, Example 1 above). To observe the development of these cells and the potential importance of the CD5 cell surface marker in the selection process we analyzed μNR CD5−/− animals. The majority of peritoneal cells were found in the conventional lymphocyte gate (www.flowjo.com; 19, 20) with the lack of CD5 not altering the B220+ B cell population (
Spleenless (Hox11−/−) mice are known to lack most of the peritoneal B-1a cell population (66). Accordingly, we found a decrease in the percentages of CD5+ B cells in the peritoneum of those mice. Interestingly, in μNR Hox11−/− the CD5 B cell levels were as high as in μNR or normal mice (
Although the spleen plays a major role in disease protection, and splenectomized patients and mice respond poorly to some immunizations, this was not seen in Hox11 mice (71). Flow cytometry confirmed that developmental progression and cell levels in the bone marrow, from the preB to the immature stage, are quite similar between Hox11−/− and normal mice although moderate variations, mostly small reductions, do occur (
A detailed comparison of antibody levels found in the various knock out and transgenic mouse lines is given in
Autonomous Expression of a HCD μ Transgene without L-Chain
The finding that μ H-chains lacking CH1 and CH2 were present on the cell surface without L-chain raised the question whether other truncated H-chains can be independently expressed or need the presence of a conventional L-chain during development. In ΔVμ transgenic mice the HCD-like human μ polypeptide without VHDJH (
To ascertain whether ΔVμ polypeptides can sustain B cell development in the absence of L-chain breeding to homozygosity was established in the L−/− background. Serum analysis showed that the levels of H-chain-only IgG in ΔVμ L−/− mice are similar to those found in L−/− mice (
A comparison of novel mouse lines with altered lymphocyte populations and silenced Ig genes, revealed that developmental processes without L-chain are adequately maintained if a truncated μ H-chain reaches the cell surface. The finding complements the reports that full length μ H-chains, deposited on the cell surface without surrogate or conventional L-chain, fail to secure B cell maturation (58-61). In camelids, H-chain-only IgG is produced from particular Cγ genes with seemingly conventional CH1 exons not included in the transcribed RNA (29, 49). In lower vertebrates H-chain dimers have been recognized that lack a classical CH1-type domain, important to provide the cysteine residue for the disulphide linkage with L-chain (11, 12). Expression of truncated single-chain IgM or IgG in transgenic mice established that the lack of CH1 is essential to secure B cell maturation (27, 28). The adaptations found in naturally produced mouse H-chain-only IgG in L−/− animals are similar to single chain Ig in camelids and cartilaginous fish (see Example 1). Surface expression of this novel BCR without L-chain appears to include the co-receptor molecules, unambiguously shown for Igβ in
Chaperone-interaction is part of the quality control machinery in the ER with BiP providing one of several partners that associate with the Ig H-chain before interaction with L-chain and disulphide-linkage (72). In the absence of L-chain BiP remains bound to CH1 (55) and upon disassociation other members of the H-chain-BiP-chaperone complex such as the most abundant GRP94 (54, 72) may complete the protein folding reaction. A high ratio of occupied BiP may activate UPR-control genes (21, 53) and it is conceivable that the inability of H-chain-only Ig to associate with BiP may prevent H-chain degradation. Alternatively, the synthesis level of truncated H-chain may exceed the degradation capacity. It may be possible to test this and preliminary evidence in older mice suggests that μNR L−/− mice have a tendency to generate Russell bodies, an Ig oligomerisation aggregate (Mattioli et al., 2006). Nevertheless, an H-chain lacking one or two complete domains appears to retain the capacity for correct folding and disulphide linkage formation, which secures transport and release from the ER. This reiterates the remarkable stability of a basically incomplete H-chain polypeptide and suggests that chaperones recognize primarily structures within intact domains, thus permitting the transport of proteins with undamaged but variable numbers of subunits. A shorter or truncated H-chain may simply be processed equivalent to the L-chain, which can be expressed at a substantially higher level than full length H-chain in the same cell (74).
Since plasma cells can produce exclusively H-chain transcripts of reduced size in the spleen (see Example 1) it was unexpected to find that H-chain-only Ig levels were secured in spleen-less Hox11−/− L−/− mice. However, B cell maturation may be similar for conventional and H-chain-only antibodies; starting in the bone marrow, followed by migration and maturation in the periphery, and the colonization of secondary lymphoid organs. This would explain why the occurrence of H-chain IgM does not appear to establish the malignant phenotype seen in human HCD. The structural similarities between H-chain antibodies and HCD drew attention to the unresolved issue whether a functional L-chain locus is implicated in the release of HCD protein. Comparing the expression of ΔVμ and μNR polypeptides in the L−/− background established that cellular exclusion is L-chain independent for both types of truncation, a lack of CH1 or in the case of DVμ and presumably other HCD proteins, deletion of VHDJH. In the DVμ mice transcripts lacking CH1 were not found, although sometimes a few residues are missing at the leader-CH1 splice junction identified by RT-PCR (62). Therefore it seems possible that a protein lacking VDJ can escape BiP-dependent retention instead of raising synthesis levels to exceed the degradation capacity.
The finding that the CD5 transmembrane glycoprotein is in association with IgM on the cell surface (75) may provide information regarding the differentiation potential of μNR cells expressing an incomplete IgM. CD5 is seen as a negative regulator of B-1a cell activation and CD5− B-1 cells show reduced apoptosis upon BCR ligation, which led to the suggestion that induction of CD5 by autoantigen may be a mechanism to avoid undesired specificities (76, 77). As monovalent IgM from μNR mice is unable to recognize antigen, the protective function of CD5 may not be utilized compared with animals producing conventional IgM. CD5− cells expressing such incomplete IgM appear to have an advantage in early development, possibly because of a more favorable proliferation signal, although disadvantages may arise after class-switching when conventional, fully functional isotypes are produced in these animals (17). Shortcomings may be revealed upon immunization and CD5 expression could be important for the selection of high affinity antibodies (78).
In summary, our analysis of mouse lines with compromised BCR show how robust and versatile antibody production is in the mammalian immune system. H-chain-only Ig expression in mice is comparable to relatively recent adaptations in camelids (13) and expression of single H-chains in cartilaginous fish, which may be a remnant of the primordial antibody structure that preceded the heterodimeric association of H- and L-chains (11). Cellular transport and release is accomplished because chaperone-recognition, which determines processing, retention and degradation, does not recognize H-chain IgM lacking distinct domains—CH1 and CH2 in μNR and VDJ in ΔVμ—as a misfolded protein. This secures surface deposition, which initiates feedback signals to progress B cell development leading to H-chain-only antibody secretion.
In healthy individuals single Ig heavy chains cannot be released from the cell because intracellular transport of Ig is only achieved upon correct folding and assembly in the endoplasmic reticulum (ER). A single H-chain of any isotype is chaperoned by association with the H-chain binding protein, BiP or grp78 (83), which is then displaced by L-chain, allowing translocation to the cell surface or secretion. However, the release of single chain IgA is observed in human αHCD. In this case, removal of VH and/or CH1 exons precludes BiP association, facilitating translocation and secretion.
In this example, we show that L−/− mice surprisingly generate a novel class of H-chain only Ig with covalently linked α-chains, not identified in any other healthy mammal. Also unexpectedly, diverse H-chain-only IgA can be released from B cells at levels similar to conventional IgA and is found in serum, milk and saliva. Surface IgA without L-chain is expressed in B220+ spleen cells, which exhibited a novel B cell receptor and suggested that associated conventional differentiation events occur. To facilitate the cellular transport and release of H-chain-only IgA, chaperoning via BiP-association seems to be prevented as only α-chains lacking CH1 are released from the cell. This appears to be accomplished by exon deletion as a result of imprecise class-switch recombination, which removes all or part of Cα1 at the genomic level.
The derivation of Igκ and Igλ deficient (L−/−, with Cκ disrupted by neo insertion and a ˜120 kb region from Cλ2 to Cλ1 removed by targeted integration and Cre-IoxP deletion), C deletion (CΔ, lacking a ˜200 kb region from Cμ to 3′ of Cμ removed by targeted integration and Cre-IoxP deletion) and μMT mice has been described (7, 45, 6). L−/− and μMT animals were crossbred to homozygosity. Mice ranging from 2 to 14 months old were analysed.
Serum antibodies were analyzed as described (46) on Falcon plates coated with 15 μg/ml anti-mouse IgA (Sigma M8769). Biotinylated detection antibody, anti-mouse IgA (Sigma B-2766), was used at a 1:300 dilution, followed by incubation with 1:300 streptavidin biotinylated horseradish peroxidase (HRP) (Amersham RPN 1051V). Antibodies in milk, saliva, urine and faeces were analyzed in the same way. For this a known mass (weight/volume) of faeces or milk (taken from the stomachs of pups) sample was dissolved in an equivalent volume (50-100 μl) of phosphate-buffered saline (PBS). Saliva from swabs was also taken into PBS (50 μl).
Serum Ig was captured on anti-mouse Ig(μ, γ and α H-chain specific; SouthernBiotech 1010-01)—or, for mass-spectrometry, anti-mouse IgA (Sigma)-coupled Sepharose, separated on Ready-Gels (Bio-Rad) under reducing or non-reducing conditions and transferred to nitrocellulose membranes as described (27). Filters were incubated with biotinylated (B10) anti-mouse antibodies specific for IgA (Sigma) or Igκ (Zymed) and λ (BD Pharmingen) L-chain. This was followed by incubation with streptavidin biotinylated HRP (Amersham) and visualization of bands using SuperSignal West Pico chemiluminescent substrate (Pierce, Ill.). Protein MW standards were supplied by Bio-Rad.
For analysis by mass-spectrometry Coomassie-stained bands were destained, reduced, carbamidomethylated and digested overnight with 10 ng/μl Sequencing Grade Modified Trypsin (Promega) in 25 mM NH4HCO3 at 30° C. The resulting peptide mixtures were separated by reversed-phase liquid chromatography as previously described (86). Proteins were identified by database searching of the mass spectral data using Mascot software (Matrix Science).
Multi-color analyses and sorting were carried out on a BD FACSAria. For analysis of IgA-positive cells, spleen cell suspensions were prepared and cells were stained with anti-mouse IgA-BIO (Sigma), CD45R (B220)-allophycocyanin (APC), CD90-FITC and F4/80-FITC (BD Pharmingen). This was followed by incubation with PE-Cy5.5-conjugated streptavidin (eBioscience). FlowJo software was used for the analysis.
For sorting of plasma cells for genomic DNA analysis, spleen cells were stained with FITC-conjugated anti-CD45R and BIO-conjugated anti-CD138 (syndecan-1) (BD Pharmingen) followed by incubation with PE-Cy5.5-conjugated streptavidin (eBioscience).
RNA was isolated from tissue or cells using Trizol (Gibco-BRL) and reverse-transcribed at 42° C. with Omniscript (Qiagen) or Bioscript (Bioline) reverse transcriptase. PCR reactions with JH and Cα3 primers were set up using KOD Hot Start DNA polymerase (Novagen), at a final MgSO4 concentration of 0.8 mM. Primers are listed in Table 5 below. The cycling conditions were 94° C. for 2 min, 40 cycles of 94° C. for 15 sec, 59° C. for 30 sec, 72° C. for 15 sec, followed by 72° C. for 10 min; the β-actin control for semi-quantitative PCR required 26 cycles at an annealing temperature of 61° C. RT-PCR products were either cleaned up (DNace Quick Clean, Bioline) and sequenced directly or cloned by adding a 3′ A overhang and using a TA Cloning Kit (Invitrogen).
PCR reactions with Cα3 and degenerate VH primers (Table 5) were set up as above, but annealing temperatures of 58° C. (J558, VH7183 and VGAM) or 52° C. (Vgen) were used, with a ramp speed of 1° C./sec. PCR products were purified from an agarose gel using the Wizard SV Gel PCR Clean-Up System (Promega) before cloning and sequencing. VH, D and JH genes were identified using the IMGT database (http://imgt.cines.fr). Agarose gels were run with size markers in by (100 by DNA ladder, Invitrogen).
Genomic DNA was prepared and analyzed by long range PCR as described previously (86). A first round PCR of 20 cycles from JH4L to Cα2L1 was followed by a nested second round PCR of 25 cycles from 3′ Eμ to Cα2L2 (primers listed in Table 5). Any bands obtained were cloned as described above or picked from the gel and re-amplified and sequenced.
ELISA discovered IgA titers in serum from L−/− mice, which in some cases were very similar to the level of conventional IgA found in normal mice (
To determine the molecular weight of the α H-chains, Western blot analysis was performed on Ig separated under reducing and non-reducing conditions (FIG. 13B,C). This shows α H-chains of ˜46 kDa, which is ˜15 kDa or one domain shorter than conventional α chains (
The production of α H-chain transcripts in different tissues was compared by semi-quantitative RT-PCR (
The analysis was extended to gain information about the VH gene repertoire of H-chain-only antibody transcripts. Amplification with different VH family oligos (J558, VGAM and V7183) identified strong bands of ˜880 bp, representing α H-chains encompassing VH-D-JH-CH2-CH3 but not CH1 (
Analysis of the VH domain showed diverse D and JH rearrangement with the addition of non-encoded residues at the junctions and extensive alterations by hypermutation (
To further investigate whether antibody specificities could potentially be selected we stained the surface of spleen cells with anti-IgA. As can be seen in
The varied H-chain-only IgA titer in L−/− mice prompted a detailed analysis of age-related and environmental constrains which could drive expression. The questions we addressed were: Do older animals produce higher Ig levels, and does an open or closed, pathogen-free, animal facility bias the expression? A comparison of IgA levels at 100-fold serum dilution is presented in
In μMT C57BI/6 animals, IgA is expressed at various levels and seemingly independent of IgM and IgD (92). To test whether H-chain-only IgA can be expressed independently we crossed L−/− mice with μMT animals to homozygosity. As shown in
The protective function of IgA plays a central role in mucosal immunity, and is often the first point of contact between the antigen and the adaptive immune system (81). IgA is also abundant in secretions, including milk and colostrum, and provides a vital source of neonatal immunity (93). To gain information as to whether H-chain-only IgA can fulfill these roles, we analyzed milk, saliva, urine and faeces from L−/− mice by ELISA (
A lack of the CH1 exon, identified in protein and transcriptional analyses of H-chain-only IgA, could be the result of either alternative splicing of RNA transcripts or genomic alteration during B cell maturation. We have looked at whether genomic deletions produce Cα regions devoid of CH1 by employing a long range PCR approach using sorted syndecan-positive plasma cells as described recently (86). Class-switch recombination from Cμ [via Cγ] to Cα retains only the last C gene and juxtaposes the rearranged VHDJH less then 10 kb upstream of Cα (94).
Serum IgA from two L−/− mice was purified by binding to anti-mouse IgA-conjugated sepharose, and size separated by SDS-PAGE, as can be seen in
For the identification of J chain, serum IgA from 5 L−/− mice was separately captured by binding to anti-mouse IgA-conjugated sepharose and directly analysed by mass-spectrometry. This resulted in the identification of α-chain and J chain peptides denoted by underlining the region in the J chain sequence from Yagi et al. (J. Exp. Med. 155:647, 1982):
KKCDPVEVEL
LTPDSCYPD
Further study of L-chain deficient mice has revealed a new type of antibody, H-chain-only IgA, which is released from the cell and surface expressed. There are no examples of the occurrence of this isotype in Tylopoda or camelids, which produce H-chain-only IgG, or in elasmobranchs (sharks, skates and rays), where H-chain-only antibodies can comprise a variable number of Cμ domains (50, 88). A common feature of murine H-chain-only IgA, as well as other naturally occurring H-chain antibodies, is the lack of a typical CH1 domain. As a result the shortened nascent-translated H-chain cannot form an association complex with the H-chain binding protein BiP as interacting CH1 residues are lacking (83, 30). The immediate advantage is that α H-chain without CH1 secures unhindered transport through the ER leading to surface deposition and H-chain-only antibody secretion (86, 95). Unexpectedly, H-chain IgA is remarkably stable, degradation seems to be prevented, and protein levels are sizeable, in some cases almost reaching conventional IgA levels in the mouse. Flow cytometry and RT-PCR identified spleen lymphocytes as a major source of α H-chain transcripts lacking CH1, which is in agreement with the recent findings of short γ transcripts in syndecan+ plasma cells. In addition, long-range PCR using DNA from sorted spleen cells identified prominent deletions in which the switch sequence and part or all of Cα1 was removed, which is similar to findings for murine H-chain-only IgG (86). However, the expression level of H-chain-only IgA and the abundance of a transcripts lacking CH1 as a dominant band in RT-PCR amplifications suggest that expression of this particular isotype is much more readily achieved. This may be due to a large number of staggered consensus repeats of the α switch region and proximal control elements such as the 3′ enhancer downstream of Cα1, a combination which may intrinsically favour aberrant switching (94, 96).
Our analysis of genomic DNA from normal mice also revealed the presence of products lacking Cα1, indicating that the mechanisms leading to heavy-chain-only antibody production exist in the normal situation (when L-chains are expressed); this is supported by the presence of a weak lower band in some RT-PCR analyses (
It has been reported that IgA may be expressed independently of IgM or IgD in early ontogeny, which could be an evolutionary primitive system that does not rely on class-switching from μ to a downstream isotype(92). As our results are consistent with the notion that H-chain-only IgA is produced and secreted by the same B cell subset as conventional IgA, we asked whether H-chain-only IgA can be expressed in the μMT background, which provides a B cell block due to a lack of surface IgM production (6). This does not seem to be the case and no H-chain IgA or any other isotype has been detected in μMT L−/− animals (see
In camelids, the VHH genes used in H-chain-only antibodies often contain specific alterations such as hallmark amino acids or an extended CDR3 region, both to compensate for the lack of L-chain, and to prevent L-chain association in a system in which both H-chain-only and conventional antibodies are produced (97, 40, 41, 42). Comparison of mouse α H-chain VHs with camelid VHHs did not, however, reveal the presence of similar alterations. This matches the finding with mouse γ H-chain VHs and reflects the fact that murine VHs have not been selected in evolution to be optimal for H-chain-only antibody production. However, it also suggests that there are fewer restrictions in the sequence of the mouse variable region; this is probably due to the complete absence of the L-chain in these mice. Therefore, murine H-chain IgA and similar H-chain only IgA binding molecules of the invention could be structurally different from the configuration of camelid H-chain IgG, and it is conceivable that two antigen-binding moieties, each contributed by a H-chain, may associate to form a single antigen-binding site. Indeed, certain VH gene sequences may be advantageous to permit different formats and the capacity of VH domains to dimerise spontaneously is not unusual (98).
Possible configurations of H-chain-only IgA according to the invention are illustrated in
The structural differences between H-chain-only and conventional IgA raised the question of whether the truncated polypeptide, without L-chain, would be recognized by the polymeric Ig receptor (or secretory component), allowing its release from mucosal surfaces. This receptor is produced in epithelial cells separate from plasma cells secreting serum IgA. Its specificity for polymeric Ig (101) implied that the level of IgA secretion into external fluids would be lower in L−/− mice as the extent of IgA oligomerization is reduced. This is indeed the case (see
Previously, production of H-chain-only IgA had only been observed in the context of disease. In L−/− mice spontaneous expression of H-chain IgA clearly differs from human αHCD, because the animals appear healthy with normal life expectancy in a pathogen-free environment. No invasion of plasma cells has been observed, which would cause lymphomas characteristic of HCD (84). Regular features associated with HCD involve deletions and insertions in the rearranged VH genes (43), neither of which have been found in VHDJH sequences from L−/− mice. However, changes to permit cellular transport are common in both systems. In L−/− mice the absence of L-chain leads to the selection and subsequent expansion of cells producing mutant α H-chains, which have lost the use of CH1.
Although the present invention has been described with reference to preferred or exemplary embodiments, those skilled in the art will recognize that various modifications and variations to the same can be accomplished without departing from the spirit and scope of the present invention and that such modifications are clearly contemplated herein. No limitation with respect to the specific embodiments disclosed herein and set forth in the appended claims is intended nor should any be inferred.
All documents cited herein are incorporated by reference in their entirety.
This application claims priority from and the benefit of U.S. Provisional Application No. 61/131,195, filed Jun. 6, 2007 and of U.S. Provisional Application No. 61/137,502, filed Jul. 30, 2008, each of the applications identified above is incorporated by reference herein for all purposes.
| Number | Date | Country | |
|---|---|---|---|
| 61131195 | Jun 2008 | US | |
| 61137502 | Jul 2008 | US |
