ENHANCED PRODUCTION OF IMMUNOGLOBULINS

Abstract

The present invention provides cells, transgenic animals, including transgenic mammals and particularly rodents, comprising engineered immunoglobulin alleles. Mutations in the alleles are designed to compromise allelic exclusion and have potential to be exploited for the isolation of bispecific antibodies.

Claims

1. A genetically modified rodent with compromised immunoglobulin heavy chain gene allelic exclusion, the genetically modified rodent comprising a genome comprising a first allele comprising a first immunoglobulin heavy chain locus and a second allele comprising a second immunoglobulin heavy chain locus, wherein the first and second immunoglobulin heavy chain loci of each allele comprise unrearranged V.sub.H, D and J.sub.H gene segments followed by a C.sub.H exon that is mutated such that heterodimerization of the encoded heavy chains is favored over homodimerization.

2. The genetically modified rodent according to claim 1, wherein the mutated C.sub.H exon comprises a C, C or C exon with a mutated C.sub.H3 domain that favors heterodimerization of the encoded heavy chains over homodimerization.

3. The genetically modified rodent according to claim 1, wherein the mutated C.sub.H exon comprises a C exon with a mutated C.sub.H3 domain that favors heterodimerization of the encoded heavy chains over homodimerization.

4. The genetically modified rodent according to claim 3, wherein the mutations in the C.sub.H3 domain of the first allele are selected from D276K, E233K, and Q234K and the mutations in the C.sub.H3 domain of the second allele are selected from K286D, K269D, and T247D.

5. The genetically modified rodent according to claim 4, wherein the first and second heavy chain allele comprise C1 exon sequences shown in SEQ ID NOs: 1 and 2.

6. The genetically modified rodent according to claim 1, wherein the mutated C.sub.H exon comprises a C or C exon with a mutated C.sub.H4 domain that favors heterodimerization of the encoded heavy chains over homodimerization.

7. The genetically modified rodent according to claim 1, wherein the immunoglobulin heavy chain locus of the first and second alleles lack exons encoding C.sub.H1 domains.

8. The genetically modified rodent according to claim 1, wherein endogenous C.sub.H have been deleted and replaced with C.sub.H exons that are mutated such that heavy chain heterodimerization is favored over homodimerization.

9. The genetically modified rodent according to claim 1, wherein the first and second immunoglobulin heavy chain loci comprise V.sub.H, D and J.sub.H genes comprising human coding sequences and rodent regulatory sequences.

10. The genetically modified rodent according to claim 1, wherein the rodent is a mouse or a rat.

11. Primary B cells, immortalized B cells, or hybridomas from the genetically modified rodent according to claim 1.

12. Primary B cells, immortalized B cells, or hybridomas according to claim 11, that express two functional heavy chains per cell and one light chain.

13. Primary B cells, immortalized B cells, or hybridomas according to claim 11, that co-express two or more different antigen receptors per cell and/or a bispecific antigen receptor.

14. Primary B cells, immortalized B cells, or hybridomas from the genetically modified rodent according to claim 7, wherein the B lymphocytes express heavy chain only antibodies.

15. An immunoglobulin heavy chain gene from the genetically modified rodent of claim 1.

16. A part or whole immunoglobulin protein encoded by the immunoglobulin heavy chain genes of claim 15.

17. A method of producing bispecific antibodies comprising immunizing the rodent according to claim 1 with two different antigens.

18. The method according to claim 17, wherein the two antigens are injected simultaneously

19. The method according to claim 17, wherein the two antigens are injected sequentially.

20. The genetically modified rodent of claim 1, which when injected with two different antigens simultaneously, or with one antigen followed by a second different antigen, generates B lymphocytes co-expressing two or more different antigen receptors per cell and/or a bispecific antigen receptor which recognize the two different antigens.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0027] FIG. 1 illustrates two typical alleles at the heavy chain locus featuring variable gene segments prior to VDJ recombination.

[0028] FIG. 2 illustrates modifications to the heavy chain alleles for permissive bispecific antibody production from the concurrent expression of two heavy chain alleles.

[0029] FIG. 3 illustrates the constant region locales of the heavy chain locus featuring exons for the expression of different antibody classes.

[0030] FIG. 4 illustrates modifications to the heavy chain alleles for bispecific antibody production by sequential heavy chain expression.

[0031] FIG. 5 illustrates alternative modifications to the heavy alleles for bispecific antibody production by sequential heavy chain expression.

[0032] FIG. 6 illustrates modifications to the heavy chain alleles for inducible bispecific antibody production.

DEFINITIONS

[0033] The terms used herein are intended to have the plain and ordinary meaning as understood by those of ordinary skill in the art. The following definitions are intended to aid the reader in understanding the present invention, but are not intended to vary or otherwise limit the meaning of such terms unless specifically indicated.

[0034] The term transgene is used herein to describe genetic material which has been or is about to be artificially inserted into the genome of a cell, and particularly a cell of a vertebrate host animal.

[0035] Transgenic animal is meant a non-human animal, usually a mammal such as a rodent, particularly a mouse or rat, although other mammals are envisioned, having an exogenous nucleic acid sequence present as a chromosomal or extrachromosomal element in a portion of its cells or stably integrated into its germ line DNA (i.e., in the genomic sequence of most or all of its cells).

[0036] A vector includes plasmids and viruses and any DNA or RNA molecule, whether self-replicating or not, which can be used to transform, transduce or transfect a cell.

[0037] Cell surface refers to the plasma membrane of the cell, i.e., that part of the cell most directly exposed to extracellular spaces and available for contact both with cells and proteins in the extracellular (including intercellular) space.

[0038] A bispecific antibody is one that comprises two physically separable antigen-binding surfaces which differ from each other in their antigen specificity. Normal antibodies have two physically separable antigen-binding surfaces that are structurally identical and thus have the same antigen specificity. A preferred version of a bispecific antibody is one that resembles a normal IgG antibody molecule with two physically separable antigen-binding surfaces, but instead of these surfaces being structurally identical, they differ from each other. In the context of this invention, both of these surfaces may be comprised of the same heavy chain protein but they would differ from each other in the light chain proteins they comprise. Alternatively, the two surfaces may be comprised of the same light chain protein but they would differ from each other in the heavy chain proteins they comprise.

[0039] As used herein, productive rearrangement is a VDJ or VJ rearrangement that is in frame and enables variable region domain translation. The variable domain of a heavy chain or light chain is considered functional if it can be expressed in-frame with the downstream constant region exons(s). A heavy chain or light chain protein translated from a productive VDJ or VJ rearrangement, respectively, is referred to as functional if it can be expressed on the cell surface.

[0040] An immunoglobulin allele described herein refers to a chromosome segment at the heavy chain or light chain locus that may include the variable gene segments, an intronic enhancer, constant regions genes, and other sequences of endogenous or exogenous sources.

[0041] Allelic exclusion refers to the fact that the vast majority of B cells in vertebrate species such as rodents or humans carry a productively rearranged heavy chain gene on only one of two homologous autosomes. Similarly, allelic exclusion at light chain loci would refer to an analogous scenario. In a more general sense, allelic exclusion applies whenever productive V(D)J rearrangement at any heavy or light chain locus inhibits further rearrangement of other heavy or light chain V(D)J gene segments, respectively, no matter where their chromosomal location. For example, if two or more sets of heavy chain VDJ linkage groups are inserted in the same chromosome, productive rearrangement at one of the heavy chain linkage groups prevents further V(D)J rearrangement at any of the other heavy chain linkage groups. The same principle applies to light chain linkage groups. In principle, this type of allelic exclusion would occur by the same mechanism as conventional allelic exclusion

[0042] Allelic inclusion refers to a loss of allelic exclusion, and thus, to the ability of B cells to produce two or more functional heavy chain variable domains and/or two or more functional light chain variable domains.

[0043] A genomic locale is any region of the genome, preferably a gene, which is associated with one particular functional aspect. The term locale is used here to refer to parts of immunoglobulin loci. For example, it can refer to that part of an immunoglobulin locus that primarily contains one kind of gene segment, such as a V gene segment locale, or a D gene segment locale, or a J gene segment locale, or more broadly, the variable locale, which includes all of the V, D and J gene segments. The constant region locale, is that part of an immunoglobulin locus that contains constant region exons.

DETAILED DESCRIPTION OF THE INVENTION

[0044] The practice of the techniques described herein may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant techniques), cell biology, biochemistry, and sequencing technology, which are within the skill of those who practice in the art. Such conventional techniques include polymer array synthesis, hybridization and ligation of polynucleotides, and detection of hybridization using a label. Specific illustrations of suitable techniques can be had by reference to the examples herein. However, other equivalent conventional procedures can, of course, also be used. Such conventional techniques and descriptions can be found in standard laboratory manuals such as Green, et al., Eds. (1999) Genome Analysis: A Laboratory Manual Series (Vols. I-IV); Weiner, Gabriel, Stephens, Eds. (2007), Genetic Variation: A Laboratory Manual; Dieffenbach, Dveksler, Eds. (2003), PCR Primer: A Laboratory Manual; Bowtell and Sambrook (2003), Condensed Protocols from Molecular Cloning. A Laboratory Manual; and Sambrook and Russell (2002), Molecular Cloning. A Laboratory Manual (all from Cold Spring Harbor Laboratory Press); Stryer, L. (1995) Biochemistry (4th Ed.) W.H. Freeman, New York N.Y.; Lehninger, Principles of Biochemistry 3.sup.rd Ed., W.H. Freeman Pub., New York, N.Y.; and Berg et al. (2002) Biochemistry, 5.sup.th Ed., W.H. Freeman Pub., New York, N.Y.; Nagy, et al., Eds. (2003) Manipulating the Mouse Embryo: A Laboratory Manual (3.sup.rd Ed.) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., all of which are herein incorporated in their entirety by reference for all purposes.

[0045] Note that as used herein and in the appended claims, the singular forms a, an, and the include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to an immunoglobulin refers to one or more such immunoglobulins, and reference to the method includes reference to equivalent steps and methods known to those skilled in the art, and so forth.

[0046] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing devices, formulations and methodologies that may be used in connection with the presently described invention.

[0047] Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both limits, ranges excluding either or both of those included limits are also included in the invention.

[0048] In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention.

The Invention in General

[0049] The present invention provides methods and compositions for the rapid isolation of bispecific antibodies from animals. B lymphocytes present in the immune system are normally prevented from expressing more than one functional heavy chain and one functional light chain by a process called allelic exclusion. The certain embodiments of the present invention provide modifications to the genomic contents of animals such that allelic exclusion is incapacitated at one or more of the immunoglobulin loci in developing B cells. The methods described herein provide B cells the ability to express more than one functional V.sub.H domain and/or more than one functional V.sub.L domain per cell.

[0050] In certain embodiments, the invention implements modifications to the immunoglobulin alleles to impinge on the normal allelic exclusion processes such that an in-frame V(D)J rearrangement of one allele does not inhibit V(D)J rearrangement of the other allele. Certain embodiments also implement two general strategies to isolate bispecific antibodies from transgenic animals. The first strategy involves concurrent immunization regimens with two or more antigens of interest. The second strategy involves sequential immunizations with the different antigens of interest.

[0051] Allelic exclusion following both heavy and light chain rearrangements requires the trafficking of heavy chains to the cell surface. In a preferred embodiment, the present invention exploits the likelihood that, if improperly folded, newly synthesized heavy chain polypeptides are retained in the ER and lack the ability to mediate allelic exclusion. Following VDJ recombination of the heavy chainin addition to the requisite V.sub.H domain pairing with the surrogate light chain complexdimerization of the heavy chain polypeptides is required to achieve proper protein folding. Heavy chain homodimerization for all isotypes, except IgM and IgE, is initiated by symmetric protein interactions between two C.sub.H3 domains. IgM and IgE heavy chains initiate homodimerization via protein interactions between their C.sub.H4 domains.

[0052] In one aspect of this embodiment, the heavy chain alleles are modified to limit the extent of isotype switching, with exons encoding the C.sub.H3 or C.sub.H4 dimerization domains on both alleles being modified to be less compatible with homodimerization but complementary to each other for heterodimerization. The heavy chain polypeptides expressed from in-frame VDJ rearrangements on either allele alone inefficiently achieve proper protein folding, and therefore cannot traffic to the cell surface to mediate allelic exclusion.

[0053] In addition to the V.sub.H-V.sub.L pairing, light chain allelic exclusion requires proper protein folding of the heavy chain C.sub.H1 domains, which are prone to misfolding (see, e.g., Feige, et al., Mol Cell 34: 569-579 (2009)). To achieve proper protein folding, each heavy chain C.sub.H1 domain must associate with a constant region of the light chain (C or C). In an extended aspect of the methods of the invention, genetic modifications to the exons encoding the C.sub.H1 domains of both heavy chain alleles as well as the exons of the light chain constant region domains are implemented, such that each light chain can associate with a heavy chain expressed from only one allele and not both.

[0054] The transgenic mice harboring the aforementioned C.sub.H3 modifications, with or without the aforementioned C.sub.H1 modifications, are immunized with the two antigens of interest simultaneously. Repeated immunizations may be employed to maximize clonal expansion of B cells recognizing both antigens. After the immunization regimen has been completed, standard hybridoma or other techniques are employed to isolate B cells producing bispecific antibodies to both antigens of interest.

[0055] In another version of the embodiment, a transgenic animal such as a transgenic mouse carries engineered versions of the heavy chain alleles on both chromosomes, wherein modifications are introduced in the heavy chain locales that express the constant domains of the heavy chain molecules. Both engineered alleles are capable of undergoing VDJ rearrangement to create heavy chain diversity during B cell development. One of the engineered alleles is also capable of expressing a full-length transmembrane heavy chain protein that can generate a pre-BCR signal. The other engineered allele is disabled in this regard.

[0056] In one aspect of this embodiment, the two engineered alleles carry recognition sequences (wild-type or mutated) for one or more site-specific recombinases such as Cre or Flp. The recognition sites are placed in such a way that site-specific recombination changes the functionality of the constant domain-encoding part of the locus. That is, if the allele is capable of expressing a fully functional heavy chain protein, then site-specific recombination deprives the locus of this property. Similarly, if the allele is incapable of expressing a fully functional heavy chain protein, then site-specific recombination confers the ability to express a functional heavy chain on the locus.

[0057] The site-specific recombinase-mediated changes just summarized are accomplished either by deleting or inverting pieces of DNA in the constant domain-encoding part of the heavy chain locus on the two homologous chromosomes. In certain embodiments of the invention, site-specific recombinase-dependent loss of constant domain full functionality on one chromosome is accompanied by synchronous, or near synchronous, gain of full functionality on the other chromosome. In a favored aspect, expression of this site-specific recombinase is under the control of a promoter from a gene that is induced during B cell activation such as C1 or Aicda (activation-induced deaminase).

[0058] The transgenic mice just described are immunized with an antigen allowing for clonal expansion of B cells expressing antibody molecules specific for the antigen. Because of the heavy chain gene modifications they carry, the antibodies specific for this antigen are comprised of heavy chains encoded by rearranged versions of only one of the two heavy chain alleles; namely, the allele defined by full functionality in its constant domain-encoding part. The other allele lacks such functionality and because of this will not encode heavy chains capable of participating in the signaling process necessary for antigen-specific clonal expansion. Repeated immunizations may be employed to maximize clonal expansion and antigen-specific antibody diversity.

[0059] After the immunization regimen has been completed, site-specific recombination is induced resulting in a switch of heavy chain constant domain functionality from one chromosome to the other. As a result of this switch, the alleles encoding the heavy chains in antibodies specific for the antigen used in the immunization are deprived of constant domain full functionality. At the same time, or near to it, the alleles that have been previously deprived of constant domain full functionality now gain this functionality. Through this switch in functionality, cells that participate in clonal expansion in response to the antigen used in the immunization regimen gain expression of new heavy chain proteins.

[0060] Subsequent to the induced site-specific recombinase-dependent switch just described, the mice are immunized with a second antigen. Clonal expansion in response to the second antigen depends on antibodies comprised of heavy chains encoded by the second allele; i.e., the one that gains constant domain functionality due to the induced site-specific recombinase event. As in the first case, the second immunization may be repeated to maximize clonal expansion and antigen-specific antibody diversity.

[0061] The clonally expanded cells expressing antibodies specific for the second antigen include those that have previously been involved in clonal expansion in response to the first antigen. During the second clonal expansion, these cells do not express the heavy chain proteins that have specificity for the first antigen but instead express different heavy chain proteins from the second allele. The heavy chain proteins they express that contribute specificity for the second antigen are encoded by the heavy chain allele that gained constant domain full functionality as a consequence of the induced site-specific recombination event.

[0062] After the second immunization regimen has been completed, hybridoma or other techniques are employed to isolate B cells specific for both the first and second antigens. A second site-specific recombinase event may be induced to restore full functionality on the allele that carries antigen specificity for the first antigen. For example, expression of the second site-specific recombinase may be under the control of an inducible system, such as those inducible by Tamoxifen or doxycycline. Through this modification, the capacity of the cells to express two different antibody molecules may be assessed: one specific for the antigen used in the first immunization regimen, and the other specific for the antigen used in the second immunization regimen.

[0063] Following the second site-specific recombination, the assembled VDJ exon on each heavy chain allele is now expressed with a protein, or a protein domain, which is a complementary half of a heterodimer (termed a heterodimerizer). In a favored aspect, the heterodimerizers are mutant IgG1 alleles, in which the fourth exons that encode the C.sub.H3 domains of both alleles are mutated such that they promote heterodimerization with each other and suppress homodimerization. Alternatively, the heterodimerizer pair may be non-immunoglobulin proteins such as c-Fos and c-Jun (which physiologically heterodimerize to form the AP-1 transcription factor), leucine zippers, or similar proteins that form heterodimers.

[0064] In another embodiment of the invention, rather than allelic exclusion being compromised at the heavy chain locus, it is instead compromised at a light chain locus. In this version of the invention, heavy chain allelic exclusion is normal. The light chain allelic inclusion version of the invention is conceptually similar to that of the heavy chain allelic inclusion version, featuring analogous modifications in the constant domain-encoding part of the relevant homologous light chain genes. It is exploited using a similar double immunization scheme combined with an appropriately-staged inducible site-specific recombination step. Bispecific B cells are identified and/or isolated in a similar fashion to what has been described for the heavy chain allelic inclusion version of the invention. This light chain allelic inclusion version of the invention yields bispecific antibodies each comprised of two light chain proteins and one heavy chain protein, whereas the heavy chain allelic inclusion version yields bispecific antibodies each comprised of one light chain protein and two heavy chains.

[0065] In an alternative embodiment, strategies analogous to those just described for heavy chain allelic inclusion are implemented to generate B cells that produce bispecific antibodies consisting of heavy chains only. In this embodiment, exons encoding the C.sub.H1 domains are removed from both heavy chain alleles. The C.sub.H1-less polypeptides expressed from each heavy chain allele should exhibit little, if any, dependence on the presence of light chains or surrogate light chains for proper protein folding as well as trafficking to the cell surface (see, e.g., Feige, et al., Mol Cell 34: 569-579 (2009)). The variable gene segments of both heavy chain alleles may be derived from animals that naturally express single-chain antibodies, such as camelids, or from other animals with or without modifications to improve the thermo-stability of the V.sub.H domains when expressed without light chains.

[0066] In one version of this embodiment, the heavy chain alleles are further modified to limit the extent of isotype switching, with exons encoding the C.sub.H3 or C.sub.H4 dimerization domains on both alleles being mutated to be less compatible with homodimerization but complementary to each other for heterodimerization. The C.sub.H1-less heavy chain polypeptides expressed from either mutant allele alone should not traffic to the cell surface to mediate allelic exclusion because the mutations suppress efficient heavy chain homodimerization. Therefore, successful B cell development depends on the concurrent expression of C.sub.H1-less heavy chains from both mutant heavy chain alleles.

[0067] Transgenic mice lacking the aforementioned C.sub.H1 domain-encoding exons and harboring the aforementioned C.sub.H3 modifications are immunized with the two antigens of interest simultaneously. Repeated immunizations may be employed to maximize clonal expansion of B cells recognizing both antigens. After the immunization regimen has been completed, standard hybridoma or other techniques are employed to isolate B cells producing bispecific antibodies to both antigens of interest.

[0068] In an alternative version of this embodiment, allelic inclusion of heavy chains lacking C.sub.H1 domains is achieved via sequential expression of each mutant heavy chain allele in an inducible manner analogous to the heavy chain allelic inclusion schemes described in the preceding embodiment. The heavy chain alleles are also modified such that an in-frame VDJ rearrangement on one allele is incapacitated for allelic exclusion and preserved for expression at a later B cell developmental stage. This is similarly achieved via disrupting the open reading frame of one heavy chain allele by a DNA cassette or by inversion of one or more exons that encode the heavy chain constant region. An inducible event of site-specific DNA recombination is likewise introduced to alternate the expression of each mutant heavy chain allele. A similar double immunization scheme followed by a second inducible site-specific recombination step is employed to obtain bispecific B cells. This heavy chain allelic inclusion version of the invention yields bispecific antibodies each comprised of two heavy chains without light chains.

[0069] In other alternative embodiments, the immunoglobulin alleles that are modified for permissive bispecific antibody production by B cells are implemented in animals that are deficient of one or more signaling molecules necessary for allelic exclusion.

[0070] In certain cases, the methods introduce modifications to the immunoglobulin genes that are designed to be conducive to bispecific antibody formation, but have minimal effects on allelic exclusion. In one specific aspect, heavy chains containing modifications analogous to the well-known knob-into-hole mutations are introduced to the exons encoding the C.sub.H3 domain of the heavy chains (see, e.g., Ridgway, et al., Protein Eng 9: 617-621 (1996)). The introduced modifications do not completely suppress homodimer formation if each modified heavy chain is expressed alone. As only one immunoglobulin allele rearranges at a time, the heavy chain homodimers expressed from the first rearranged allele may retain competence for allelic exclusion. The present invention implements such immunoglobulin allele modifications, which on their own have minimal impact on allelic exclusion, in animals or cells that have impaired allelic exclusion caused by the deficiency of one or more signaling components of the pre-BCR or BCR. One example of such mutants is the mouse strain deficient of E2A (see, e.g., Hauser, et al., Journal of Immunology 192:2460-2470 (2014)).

[0071] The transgenic mice harboring the aforementioned C.sub.H3 modifications and a defective pre-BCR or BCR signaling component are immunized with the two antigens of interest simultaneously. Repeated immunizations may be employed to maximize clonal expansion of B cells recognizing both antigens. After the immunization regimen has been completed, standard hybridoma or other techniques are employed to isolate B cells producing bispecific antibodies to both antigens of interest.

[0072] FIG. 1 depicts two heavy chain alleles (101 and 102) typically found in humans and most vertebrate animals. Each heavy chain allele consists of multiple V (103), D (104), and J (105) genes upstream of the C exons that encode the constant regions of IgM (108). On each chromosome, regulatory elements such as the 5 intronic enhancer (106) and switch region (107) also exist between the J genes and the first C.sub.H exon. In the preferred embodiments, the V, D, and J gene segments of the transgenic mice are chimeric, consisting of human coding regions and mouse noncoding regions. Labeled components in the figure are as follows: Chromosomal segments of the heavy chain locus (101, 102); Variable region gene segments (103); D region gene segments (104); J region gene segments (105); Heavy chain intronic enhancer (106); Switch region (107); C exons encoding the constant regions of IgM (108).

[0073] FIG. 2 depicts an example of modifications that the present invention implements to both heavy chain alleles (201, 202) in order to render B cells permissive for allelic inclusion. Under germline configuration, each heavy chain allele consists of unrearranged V (203), D (204), and J (205) genes followed by modified C exons encoding an IgG isotype (208 and 209). In the preferred embodiments, the V, D, and J gene segments of the transgenic mice are chimeric, consisting of human coding regions and mouse noncoding regions. During B cell development, randomly selected V, D, and J genes assemble (210) to form the V.sub.H exons (211, 212). Certain codons within the C.sub.H3 exons of each heavy chain allele are mutated such that the encoded heavy chains are compatible with heterodimerization with each other (such as mutant C1, whose cDNA sequences for the two heavy chain alleles are specified at [SEQ ID No. 1 and 2]). Additionally, the modifications cause the translated heavy chain polypeptides to misfold if either allele is expressed alone. Consequently, pre-BCRs cannot form and traffic to the plasma membrane to mediate allelic exclusion, unless the mutant heavy chain polypeptides form heterodimers from the concurrent expression of both alleles. In an extended version of the embodiment, in combination with the aforementioned C.sub.H3 mutations, certain codons within the C.sub.H1 exons of both heavy chain alleles are similarly modified such that each heavy chain can be paired with a different light chain. Although this figure exemplifies the use of mutant IgG heavy chains to suppress allelic exclusion as well as to facilitate heavy chain heterodimerization, other isotypes (i.e., IgM, IgD, or IgA) could be similarly employed. Labeled components in the figure are as follows: Chromosomal segments of modified heavy chain alleles (201, 202); Variable region gene segments (203); D region gene segments (204); J region gene segments (205); Heavy chain intronic enhancer (206); Switch region (207); C exons encoding mutant constant regions of IgG (208, 209); VDJ recombination events (210); Assembled VDJ exons (211, 212).

[0074] FIG. 3 depicts the heavy chain genes on two homologous chromosomes found in rodents. (The typical human heavy chain locus is quite similar but has more constant region genes.) In this figure, the V (303) and D (304) genes of both heavy chain alleles (301, 302) are compressed (denoted by n) compared to FIG. 1 so as to emphasize the structure of the constant region locales of the chromosomes. In the preferred embodiments, the V (303), D (304), and J (305) gene segments of the transgenic mice are chimeric, consisting of human coding regions and mouse noncoding regions. IgM and IgD (308, individual C.sub.H exons not shown) are the first isotypes to be expressed by B cells. The C.sub.H exons encoding other antibody classes (309-314, individual exons not shown) exist further downstream. In the C57BL/6 mouse strain, these are C3 (309), C1 (310), C2b (311), C2c (312), C (313), and C (314). Certain mouse strains such as BALB/c have C2a instead of C2c. Except for that of C, an isotype switch region (307) is present preceding the first C.sub.H exon of each antibody class. Labeled components in the figure are as follows: Heavy chain alleles (301, 302); V genes denoted in brackets, n referring to multiple number of the gene segments (303); D genes denoted in brackets, n referring to multiple number of the gene segments (304); J gene (305); Heavy chain intronic enhancer (306); Switch region (307); C and C (308, individual exons not shown); C.sub.H exons of IgGs and other antibody classes (309-314, individual exons not shown).

[0075] Depicted in FIG. 4 are two heavy chain alleles (401, 402) after VDJ rearrangements have occurred to produce different V.sub.H exons (403, 404), as they would be found in naive B cells. On allele 401, the exons encoding C and/or C (406) are in the same sense orientation (arrow below) as the rearranged VDJ exon (403). By contrast, the C and/or C exons (412) are present in the reverse orientation (arrow below) relative to the VDJ exon (404) on allele 402. Thus, allele 402 is not capable of expressing full-length heavy chain polypeptides as its C and/or C exons are in the antisense configuration, and normal B cell development is dependent on the expression of functional heavy chains from allele 401. The inverted C and/or C exons (412) in allele 402 are immediately flanked by two oppositely oriented recognition sequences (409, 410) for site-specific DNA recombination. In allele 401, the two recognition sequences for site-specific DNA recombination (409, 410) are placed further apart, also in opposite orientation: one (409) upstream of the C and/or C exons (406), the other (410) downstream of C exons (408). During a B cell response to an immunogen (414), allele 401 undergoes isotype switching, which brings the downstream site-specific DNA recombination sequence (410) closer to its upstream counterpart (409). When expression of the site-specific DNA recombinase is induced (415), the DNA segments flanked by its recognition sequences (409, 410) on both alleles undergo irreversible inversion because the resultant recombination sites (416, 417) are no longer competent for recombination. Allele 402 is now capable of expressing full-length heavy chains, while allele 401 becomes inactivated by the inversion of its C.sub.H exons. Following immunization with a second antigen (418), allele 402 undergoes isotype switching. The invertible DNA segments of both alleles are additionally flanked by recognition sequences (411) for a second site-specific DNA recombinase, which can be introduced before or after hybridoma generation. Following excision of the intervening DNA fragments, the VDJ exons on both alleles (403, 404) are now expressed with the downstream C.sub.H exons modified to be compatible with heavy chain heterodimerization (413). Labeled components in the figure are as follows: Heavy chain alleles (401, 402); Assembled VDJ exons (403, 404); Heavy chain intronic enhancer (405); C and/or C exon(s) (406) initially in sense orientation (arrow below) relative to assembled VDJ exon (403); Switch region (407); C.sub.H exons of switched isotype (408); Recognition sequences for the first site-specific recombinase (409, the sequence of this site is in opposite orientation to the site labeled 410); Recognition sequence for the second site-specific recombinase (411); C and/or C exon(s) (412) initially in reverse orientation (arrow below) relative to assembled VDJ exon (404); Modified exons compatible with heavy chain heterodimerization (413); Antigen response followed by isotype switching (414); First site-specific DNA recombinase expression (415); Remnant DNA sequences following site-specific DNA recombination (416, 417); Antigen response to second immunogen and isotype switching (418).

[0076] FIG. 5 illustrates alternative modifications to the heavy alleles for bispecific antibody production by sequential heavy chain expression. Shown are two heavy chain alleles (501, 502) after VDJ rearrangements have occurred to produce different V.sub.H exons (503, 504), as they would be found in naive B cells. On allele 501, a DNA cassette (512) flanked by two oppositely oriented recognition sequences (509, 510) for a site-specific DNA recombinase is inserted downstream of the J genes before the exons encoding C and/or C constant domains (506). The DNA cassette (512) is in reverse orientation (arrow below) relative to the assembled VDJ exon (503) and contains one or more of the following: a splice acceptor, a ribosomal skip sequence or IRES, an open reading frame, a stop codon, or a poly-adenylation signal sequence. A similar DNA cassette (514) is also inserted at an analogous locale on allele 502, but is in the same sense orientation (arrow below) as the rearranged VDJ exon (504). Allele 502 cannot express full-length heavy chains because the DNA cassette (514) disrupts its open reading frame following the VDJ exon (504). By contrast, allele 501 can express full-length heavy chains because the inverted DNA cassette does not disrupt its open reading frame. Downstream of the IgG exons of both alleles is a recognition site (511) for a second DNA recombinase. Also on both alleles, another site for the second DNA recombinase (511) is inserted upstream of the recognition sites for the first DNA recombinase (509). During a B cell response to an immunogen (515), allele 501 undergoes isotype switching. When expression of the first site-specific DNA recombinase is induced (516), the DNA cassettes on both alleles (512, 514) undergo irreversible inversion because the resultant recombination sites (517, 518) are no longer competent for recombination. Allele 502 is now capable of expressing full-length heavy chains because the DNA cassette (514) is now in reverse orientation and no longer disrupts the heavy chain open reading frame. By contrast, the open reading frame of allele 501 is disrupted by the DNA cassette (512) in the same orientation as its VDJ exon (503). Following immunization with a second antigen (519), allele 502 undergoes isotype switching. When expression of the second site-specific DNA recombinase is introduced before or after hybridoma generation, the intervening DNA fragments (512, 514) are now excised at its cognate recognition sequences (511). Subsequently, the VDJ exons on both alleles (503, 504) are now expressed with C.sub.H exons modified for heavy chain heterodimerization (513). Labeled components in the figure are as follows: Heavy chain alleles (501, 502); Assembled VDJ exons (503, 504); Heavy chain intronic enhancer (505); C and/or C exons (506); Switch region (507); C.sub.H exons of switched isotype (508); Recognition sequences for the first site-specific recombinase (509, the sequence of this site is in opposite orientation to the site labeled 510); Recognition sequence for the second site-specific recombinase (511); DNA cassette (512) initially in reverse orientation (arrow below) relative to VDJ exons (503); Modified exons compatible with heavy chain heterodimerization (513); DNA cassette (514) initially in sense orientation (arrow below) relative to VDJ exon (504); Antigen response followed by isotype switching (515); First site-specific DNA recombinase expression (516); Remnant DNA sequences following site-specific DNA recombination (517, 518); Antigen response to second immunogen and isotype switching (519).

[0077] FIG. 6 illustrates modifications to the heavy chain alleles for inducible bispecific antibody production. In germline configuration, one heavy chain allele (601) consists of unrearranged V (603), D (604) and J (605) genes followed by the C and/or C exons (608) that are flanked by two recognition sequences for a site-specific DNA recombinase (609). On the other heavy chain allele (602), a DNA cassette (610) in sense orientation (arrow below) flanked by two recognition sequences for the same site-specific DNA recombinase (609) is inserted downstream of the V, D, and J genes. In the preferred embodiments, the V, D, and J gene segments of the transgenic mice are chimeric, consisting of human coding regions and mouse noncoding regions. The DNA cassette contains one or more of the following: a splice acceptor, a ribosomal skip sequence or IRES, an open reading frame, a stop codon, or a poly-adenylation signal sequence. Additionally, the heavy chain alleles contain the same modifications to the C.sub.H exons for heterodimerization (611 and 612) as those described in FIG. 2 (208 and 209 in FIG. 2). Following VDJ recombination events (614), successful assembly of an in-frame V.sub.H exon (615) on allele 601 results in normal expression of full-length heavy chains. By contrast, the DNA cassette (610) on allele 602 prevents any successful VDJ rearrangement (616) from being able to express full-length heavy chain polypeptides. Upon expression of a site-specific DNA recombinase gene (617), C.sub.H exons (608) on the first allele (601) as well as the DNA cassette (610) on the second allele (602) are excised from the chromosomes. VDJ exons on both alleles (615, 616) can now be expressed with downstream C.sub.H exons containing modifications conducive to heavy chain heterodimerization. Labeled components in the figure are as follows: Modified heavy chain alleles (601, 602); V genes denoted in brackets, with n referring to the presence of multiple gene segments (603); D genes denoted in brackets, with n referring to the presence of multiple gene segments (604); J genes (605); Heavy chain intronic enhancer (606); Switch region (607); C and/or C exons (608); Recognition sequences for site-specific DNA recombinases (609); DNA cassette in sense orientation indicated by arrow below (610); Mutant C1 exons (611; 612); Heavy chain 3 enhancer (613); VDJ recombination events (614); Assembled VDJ exons (615, 616); Activation of site-specific DNA recombinase (617).

Transgenic Cell Libraries

[0078] The transgenic cells of the invention may be used to produce expression libraries, preferably low complexity libraries, for identification of antibodies of interest on the surface of plasma cells. The present invention thus also includes antibody libraries produced using the cell technologies of the invention for identification of antigen-specific antibodies expressed on plasma cells.

Transgenic Animals

[0079] In specific aspects of the invention, the invention provides transgenic animals carrying engineered in heavy chain or light genes.

[0080] In certain embodiments, the transgenic animals of the invention further comprise human immunoglobulin regions. For example, numerous methods have been developed for replacing endogenous mouse immunoglobulin regions with human immunoglobulin sequences to create partially- or fully-human antibodies for drug discovery purposes. Examples of such mice include those described in, for example, U.S. Pat. Nos. 7,145,056; 7,064,244; 7,041,871; 6,673,986; 6,596,541; 6,570,061; 6,162,963; 6,130,364; 6,091,001; 6,023,010; 5,593,598; 5,877,397; 5,874,299; 5,814,318; 5,789,650; 5,661,016; 5,612,205; and 5,591,669.

[0081] In a particularly preferred aspect, the transgenic animals of the invention comprise chimeric immunoglobulin segments as described in co-pending application US Pub. No. 2013/0219535 by Wabl and Killeen. Such transgenic animals have a genome comprising an introduced partially human immunoglobulin region, where the introduced region comprising human variable region coding sequences and non-coding variable sequences based on the endogenous genome of the non-human vertebrate. Preferably, the transgenic cells and animals of the invention have genomes in which part or all of the endogenous immunoglobulin region is removed.

Use in Antibody Production

[0082] Culturing cells in vitro has been the basis of the production of numerous therapeutic biotechnology products, and involves the production of protein products in cells and release into the support medium. The quantity and quality of protein production over time from the cells growing in culture depends on a number of factors, such as, for example, cell density, cell cycle phase, cellular biosynthesis rates of the proteins, condition of the medium used to support cell viability and growth, and the longevity of the cells in culture. (See, for example, Fresney, Culture of Animal Cells, Wiley, Blackwell (2010); and Cell Culture Technology for Pharmaceutical and Cell-Based Therapies, Ozturk and Ha, Eds., CRC Press, (2006).)

[0083] The invention provides a source of B cells derived from immunization schemes in which animals are challenged with two or more antigens simultaneously, or first with one antigen and then later with another antigen. In both cases, multiple immunizations may be employed to increase antigen-specific antibody titers in individual animals. An inducible site-specific recombination step is included between the two immunization series. Subsequent to the final immunization scheme, B cells are isolated and cultured or used to create hybridomas, or used as a source of RNA for cloning immunoglobulin chain genes. The B cells or the antibody chains they contain are tested for bispecific antigen-binding properties. In the case of hybridomas, this is accomplished by screening hybridomas directly for bispecific antibodies, or for specificity for one kind of antigen and then further analyzing them for whether they carry additional rearranged immunoglobulin chain genes that confer specificity for a second kind of antigen, i.e., they have latent or expressed bi-specificity.

EXAMPLES

[0084] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent or imply that the experiments below are all of or the only experiments performed. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0085] Efforts have been made to ensure accuracy with respect to terms and numbers used (e.g., vectors, amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees centigrade, and pressure is at or near atmospheric.

Example 1. Engineered Heavy Chain Alleles Permissive for the Isolation of Bispecific Antibodies Following Simultaneous Immunization with Two or More Antigens

[0086] Transgenic mice are generated carrying two modified heavy chain alleles that lack the ability to isotype switch (as depicted in FIG. 2). Both alleles can undergo VDJ recombination. In a preferred embodiment, an IgG1 instead of IgM heavy chain is expressed during B cell development, but any other isotype including IgM may be employed. B cells expressing only IgG1 develop quite normally and respond to antigens during immunization (see, e.g., Waisman, et al., Journal of Experimental Medicine 204:747-758 (2007)).

[0087] The fourth exons of both IgG1 alleles, which encode the C.sub.H3 domains, are mutated such that they promote the formation of heavy chain heterodimers and suppress homodimerization. In preferred methods, the mutations are D276K, E233K, and Q234K on one heavy chain allele; and K286D, K269D, and T247D on the other heavy chain allele (amino acid numbering starts at the first codon of C.sub.H1). The mutations at similar positions in the human IgG1 heavy chain have been shown to promote heterodimerization and the secretion of bispecific antibodies in cell lines (see, e.g., Gunasekaran, et al., Journal of Biological Chemistry 285:19637-19646 (2010)).

[0088] During B cell development, pro-B cells that have rearranged only one heavy chain allele are blocked in development, because the mutant IgG1 heavy chains inefficiently homodimerize to form pre-BCRs. Therefore, insufficient signals are transduced to mediate allelic exclusion, and the second heavy chain allele expressing mutant C1 becomes permissive for VDJ recombination. Developing B cells can progress beyond the pro-B cell stage only when both mutant heavy chains are co-expressed and heterodimerize to form pre-BCRs.

[0089] Ultimately, mature B cells in the transgenic mice of the preferred methods harbor two functional heavy chains per cell with one light chain. The transgenic mice may be used for simultaneous immunization with two or more antigens of interest with the subsequent generation of hybridomas using standard methodologies. Hybridomas derived from the transgenic mice are permissive for the direct isolation of bispecific antibodies.

[0090] In exemplary embodiments of the heavy chain alleles for this Example 1, the V (203), D (204), and J (205) genes of both heavy chain alleles (201, 202) comprise human coding sequences with mouse regulatory sequences and are described in the co-pending application US Pub. No. 2013/0219535 by Wabl and Killeen. Sequences of the heavy chain intronic enhancer (206) as well as all downstream elements, including the heavy chain constant region genes, found in a wild-type mouse are described in LOCUS: NG_005838 (1 . . . 180,971). In this Example 1, the sequence spanning 8,608 . . . 175,134 containing the C.sub.H exons of all isotypes is deleted from both heavy chain alleles and is replaced by modified C1 exons (elements 208 and 209 in FIG. 2). Exon 4 of both elements 208 and 209 are mutated to favor heterodimerization with each other over dimerization with self. The mutant C1 cDNA sequences of both heavy chain alleles are specified at [SEQ ID No. 1 and 2], as well as the wild type cDNA sequence of the heavy chain allele [SEQ ID No. 3].

Example 2. Engineered Heavy Chain Alleles Permissive for the Isolation of Bispecific Antibodies Following Sequential Immunizations

[0091] Transgenic mice are generated carrying heavy chain alleles that can be switched on or off by site-specific DNA recombination. One of the alleles is capable of expressing full-length heavy chains after a productive VDJ rearrangement, while the other allele lacks this functionality.

[0092] Both alleles contain recognition sequences for one or more site-specific recombinases, positioned within the constant domain-encoding locale. Site-specific recombination at these sites causes an inversion of a piece of DNA in both alleles, and as a consequence of this inversion, the constant domain functionality in the alleles is changed.

[0093] Site-specific recombination confers the capacity to express a full-length heavy chain protein on the allele that initially lacked this capacity. By contrast, site-specific recombination removes this capacity from the allele that initially has it.

[0094] One version of this embodiment is depicted in FIG. 4, with the relevant site-specific recombination sequences marked as 409 and 410, and with the constant domain-encoding exons labeled as 406 and 412. The transcriptional orientation of the constant domain-encoding exons is depicted by the arrow immediately below the labeled components.

[0095] FIG. 4 shows the two alleles in their configuration after VDJ rearrangements have occurred to generate the V.sub.H exons (403 and 404). However, VDJ rearrangements of the alleles depicted in FIG. 4 could result in nonproductive gene segment joins on either allele. Similarly, the process could also result in productive rearrangements on either allele. B cells only complete their development in the bone marrow if they express full-length heavy chain molecules from allele 401, because even though allele 402 can undergo a productive VDJ recombination, it cannot express full-length heavy chains since all or part of the C and/or C exons are in the reverse orientation.

[0096] Peripheral B cells that develop in the mice carrying the alleles shown in FIG. 4 express B cell antigen receptors (transmembrane or secreted) comprised of heavy chains derived from allele 401. The light chains in these B cell antigen receptors derive from normal independent VJ rearrangements at one of their light chain loci.

[0097] Immunization of mice carrying the alleles shown in FIG. 4 result in clonal expansion of B cells expressing antibodies specific for the immunogen. Crucially, once again, these antibodies are solely comprised of heavy chains derived from allele 401. Repeated immunizations result in enhanced clonal expansion, somatic hypermutation, and isotype switching similar to what occurs in normal mice (shown as 414 in FIG. 4).

[0098] Transgenic mouse systems exist, or can be readily engineered, to permit inducible expression of particular site-specific DNA recombinases in multiple cell types including B lymphocytes. In yet another embodiment of the present methods, transgenic mice carrying the mutant alleles 401 and 402 depicted in FIG. 4 additionally harbor an inducible site-specific recombinase system, such as the tamoxifen-inducible system. Immunized mice that have made demonstrable antibody responses to the antigen used in the immunization are caused to express the relevant site-specific recombinase.

[0099] Following the expression of a site-specific DNA recombinase (415 in FIG. 4), the chromosomal segments flanked by sites for the induced DNA recombinases undergo inversion on both alleles. A resultant feature is the loss of capacity to express full-length heavy chains on allele 401. Concurrently, allele 402 gains the capacity to express full-length heavy chains.

[0100] Allele 402 is productively rearranged in some of the B cells that have undergone clonal expansion in response to the immunization. The frequency of such second allele productive rearrangements is typically much higher than is normally the case because this allele does not express full-length heavy chains during B cell development, yet it nonetheless is capable of undergoing VDJ rearrangements.

[0101] Immunization of mice carrying the newly activated allele 402 depicted in FIG. 4 results in clonal expansion of B cells expressing B cell antigen receptors specific for the antigen used in the immunization. These B cell antigen receptors (transmembrane or secreted) are comprised solely of heavy chains derived from allele 402. Repeated immunizations result in enhanced clonal expansion, somatic hypermutation, and isotype switching similar to what occurs in normal mice.

[0102] B cells specific for the antigen used in the second immunization include some that have not undergone clonal expansion in response to the first antigen. Such B cells are not a desirable source for bispecific antibodies capable of recognizing the antigens used in both of the immunizations.

[0103] However, some fraction of the B cells specific for the second antigen have been involved in clonal expansion in response to the first antigen. These B cells are an obvious source for bispecific antibodies since one of their rearranged heavy chain genes carries specificity for the first immunogen, while their other rearranged heavy chain genes carries specificity for the second immunogen. In both cases, individual B cells pair one light chain protein with both heavy chain proteins.

[0104] Hybridoma or other cloning technology may be exploited to recover B cells with specificity for the second immunizing antigen. These B cells are then analyzed to determine whether they also carry a rearranged heavy chain gene that confers specificity for the first immunizing antigen.

Example 3. Alternative Engineered Heavy Chain Alleles Permissive for the Isolation of Bispecific Antibodies Following Sequential Immunizations

[0105] The methods described here are very similar to those described in Example 2. Transgenic mice are engineered to carry two heavy chain immunoglobulin alleles that can be switched on or off by a site-specific DNA recombinase system. The two alleles are designed for sequential immunization schemes similar to those described in Example 2, and consequently feature largely the same kind of functionality in their constant domain locales. Where the alleles differ is in the inclusion of elements designed to improve the efficiency with which the desired kind of bispecific B cells are isolated.

[0106] This embodiment is depicted in FIG. 5. On one of the two engineered heavy chain alleles (allele 501), a DNA cassette (512) flanked by two recognition sequences (509 and 510) for a site-specific DNA recombinase is inserted after the heavy chain enhancer (505) but before the switch region (507) preceding the C exons (506), so that the heavy chain enhancer (505) remains in the genome after isotype switching. On the second heavy chain allele (allele 502), similar elements are inserted at an analogous position, but in the opposite orientation (arrow below). The DNA cassettes are designed to disrupt the open reading frame of the heavy chain exons when aligned in the same transcriptional orientation as the assembled VDJ exon.

[0107] In one embodiment, allele 501 consists of exons 15 and 16 from the murine integrin beta-7 (Itgb7) gene, aligned in the opposite orientation from the V.sub.H exon. Both Itgb7 exons contain a splice acceptor. Additionally, Itgb7 exon 15 harbors a stop codon, while Itgb7 exon 16 contains a stop codon as well as a poly-adenylation sequence signal. Because of the inverted transcriptional orientation, this DNA cassette does not interfere with heavy chain expression from an in-frame VDJ recombination on allele 501.

[0108] In a preferred configuration, the inserted DNA cassette in allele 502 consists of an open reading frame from a gene that provides survival, functional, or selection advantages to the B cells that have successfully assembled an in-frame V.sub.H exon from VDJ recombination. An example of such gene is the anti-apoptotic B-cell lymphoma-2 (Bcl2). The open reading frame of the advantageous gene is aligned in the same transcriptional orientation as the heavy chain mRNA. To prevent this gene from being expressed as a protein fused to the V.sub.H exon, a ribosomal skip sequence such as the 2A peptide from a picornavirus is placed between the splice acceptor and the open reading frame of the advantageous gene. The 2A peptide also ensures that the advantageous gene is only expressed in the B lymphocytes that have successfully assembled an in-frame V.sub.H exon, and not in the B cells that lack a productive VDJ rearrangement.

[0109] In one exemplary embodiment, the assembled VDJ genes (503, 504) on both heavy chain alleles (501, 502) are derived from individual gene segments comprising human coding sequences with mouse regulatory sequences and are described in the co-pending application US Pub. No. 2013/0219535 by Wabl and Killeen. All endogenous sequences downstream, including the heavy chain constant region genes, are described in LOCUS: NG_005838 (1 . . . 180,971). Sequences of the Itgb7 and Bcl2 DNA cassettes (elements 512 and 514, respectively) are specified at [SEQ ID Nos. 4 and 5] and are inserted at around position 178,000 of the locus, in between the heavy chain intronic enhancer and the switch region of the first exon C exon. In this embodiment, recognition sequences for the first site-specific DNA recombinase (elements 509 and 510) are lox66 and lox71 (see, e.g., Oberdoerffer, et al., Nucleic Acids Res 31:e140 (2003)). Also in this embodiment, recognition sequences for the second site-specific DNA recombinase (elements 511) are those used by Flp enzyme (see, e.g., McLeod, et al., Mol Cell Biol 6:3357-3367 (1986)). In this embodiment, the heterodimers (element 513 on each heavy chain allele) are mutant C1 designed to favor heterodimerization with each other over self-dimerization. The mutant C1 cDNA sequences of both heavy chain alleles are specified at [SEQ ID Nos. 1 and 2].

[0110] As in Example 2, following the first immunization, allele 501 can respond normally to the antigen because the inverted Itgb7 cassette has no effect on the transcriptional activities of the heavy chain locus. Because the second DNA cassette interrupts the heavy chain open reading frame of allele 502, no specificity for the immunogen is selected from allele 502 following the first immunization.

[0111] Upon expression of a site-specific DNA recombinase (516), the DNA cassettes on both heavy chain alleles are inverted. The open reading frame of the Itgb7 gene cassette in allele 501 is now in the same transcriptional orientation as the heavy chain mRNA. Effectively, the stop codons and poly-adenylation signal sequence of the Itgb7 gene prevents the expression of full-length heavy chains from allele 501.

[0112] By contrast, allele 502 can now express full-length heavy chains because the open reading frame of the inserted advantageous gene is no longer in the same orientation as the heavy chain mRNA. Allele 502 subsequently can respond to the immunogen in the second the immunization.

[0113] Hybridoma or other cloning technology may be exploited to recover B cells with specificity for the second immunizing antigen. These B cells can then be analyzed to determine whether they also carry a rearranged heavy chain gene that confers specificity for the first immunizing antigen.

Example 4. Alternative Engineered Heavy Chain Alleles Permissive for the Isolation of Bispecific Antibodies Following Simultaneous Immunization with Two or More Antigens

[0114] The scheme for this Example 4 is depicted in FIG. 6 and contains some elements of both Example 1 as well as Example 3. Transgenic mice are engineered to carry two modified heavy chain alleles that lack the ability to isotype switch by normal means. On one heavy chain allele (601 in FIG. 6), the C and/or C heavy chain exons are flanked by directly-oriented recognition sequences for a site-specific recombinase. Further downstream are C1 exons containing C.sub.H3 mutations for inter-heavy chain heterodimerization as described for allele 201 in FIG. 2 and Example 3, such as the mutant C1 cDNA sequences for two heavy chain alleles as specified at [SEQ ID No. 1 and 2]. Allele 601 can support normal B cell development because its open reading frame transcribed from the assembled V.sub.H exon is uninterrupted by the recognition sequences for the site-specific DNA recombinase. On the second heavy chain allele (602 in FIG. 6), a DNA cassette (610) flanked by the directly-oriented recognition sequences (609) for the same site-specific DNA recombinase is inserted downstream of the J genes (605). Thus, an in-frame VDJ assembly on allele 602 is deprived of the capacity to express full-length heavy chains because the DNA cassette (610) is designed to disrupt its open reading frame. Further downstream of these elements are the C1 exons containing complementary C.sub.H3 mutations for heterotypic association with the mutant IgG1 heavy chain encoded by allele 601.

[0115] In a preferred configuration, as in Example 3, the inserted DNA cassette in allele 602 comprises an open reading frame from a gene that provides survival, functional, or selection advantages to the B cells that have successfully assembled an in-frame V.sub.H exon from VDJ recombination. The DNA cassette also contains a splice acceptor and a 2A peptide sequence preceding the open reading frame of the advantageous gene. Crucially, the 2A peptide also ensures that the advantageous gene is only expressed in the B lymphocytes that have successfully assembled an in-frame V.sub.H exon, and not in the B cells that lack a productive VDJ rearrangement.

[0116] When expression of a site-specific DNA recombinase is introduced, the intervening DNA segments flanked by the recognition sequences for the site-specific DNA recombinase is excised from both heavy chain alleles. Subsequently, both heavy chains are now competent for full-length heavy chain expression. As described in Example 1, the two mutant IgG1 heavy chains are mutually dependent on each other for cell surface expression and secretion. Expression of either mutant heavy chain allele alone likely results in B cell death, because cell surface expression of the BCR is also required for the survival of mature B cells (see, e.g., Lam, et al., Cell 90:1073-1083 (1997)).

[0117] As in Example 1, mature B cells in the transgenic mice of this example harbor two functional heavy chains per cell with one light chain. The transgenic mice may be used subsequently for the simultaneous immunization with two or more antigens of interest. Hybridomas harboring bispecific antibodies are generated using standard methodologies.

[0118] For example in an exemplary embodiment, the V (603), D (604), and J (605) genes of both heavy chain alleles (601, 602) comprise human coding sequences with mouse regulatory sequences as described in the co-pending application US Pub. No. 2013/0219535 by Wabl and Killeen. Sequences of the heavy chain intronic enhancer (606) as well as all downstream elements, including the heavy chain constant region genes found in a wild-type mouse are described in LOCUS: NG_005838 (1 . . . 180,971). On allele 601, the sequence spanning 8,608 . . . 168,728 that contain C and C.sub.H exons of all downstream isotypes is deleted. Additionally, the C exons of allele 601 (171230 . . . 175134 of the locus) are flanked by two directly oriented standard loxP sites. On allele 602, the sequence spanning 8,608 . . . 175,134 of the locus containing the C.sub.H exons of all isotypes is deleted. An Itgb7 DNA cassette flanked by two directly oriented loxP sites is inserted at around position 178,000 of the locus. Downstream of the 3 loxP sites on both alleles are sequences of the modified C1 exons (elements 611, 612). Exon 4 of both elements 611 and 612 are mutated to favor heterodimerization with each other over self-dimerization. The mutant C1 cDNA sequences of both heavy chain alleles are specified at [SEQ ID No. 1 and 2],

[0119] The preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims that follow, unless the term means is used, none of the features or elements recited therein should be construed as means-plus-function limitations pursuant to 35 U.S.C. 112, 6.