DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Continued Examination Under 37 CFR 1.114
1. A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on June 23, 2026 has been entered.
Claims 4, 21, and 22 are now pending and being examined. Claims 4 and 22 are amended.
Claim Objections
2. Claim 4 is objected to because of the following informalities: Claim 4 recites the name of immunoglobulin genes that comprise a hyphen in the name, for example, IGHV3-3, IGHV3-4, etc. Claim 4 also uses a hyphen in the names of genes to indicate a list of genes, for example, IGHD1-6 means IGHD1, IGHD2, IGHD3, IGHD4, IGHD5, and IGHD6. The meaning of the hyphen is inconsistent in claim 4. Examiner suggests employing one meaning of the hyphen as part of the gene name and spelling out the list of genes where the hyphen is used to indicate a list. Appropriate correction is required.
New Rejection
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
3. Claim(s) 4, 21, and 22 are rejected under 35 U.S.C. 103 as being unpatentable over US Patent 10,662,256, Wabl, claiming priority to 2010; in view of US Patent Application Publication 2013/0167256, Green et al; Hwang et al (Veterinary Immunology and Immunopathy, August 2018, 202:181-190); and NCBI Canis lupus familiaris genome assembly CanFam3.1 version GCF_000002285.3 (posted online 2011).
Wabl teaches and suggests a transgenic mouse comprising a genome comprising a nucleic acid encoding canine DNA, wherein the DNA encodes or expresses canine heavy chain VH, DH, and JH (IGHV, IGHD, and IGHJ) gene segments, and Vλ, Jλ and Cλ (IGLV, IGLJ, AND IGLC) gene segments:
An embodiment of the invention provides a method for generating the transgenic rodent, said method comprising: a) integrating at least one target site for a site-specific recombinase in a rodent cell’s genome upstream of an endogenous immunoglobulin variable gene locus and at least one target site for a site-specific recombinase downstream of the endogenous immunoglobulin variable gene locus, wherein the endogenous immunoglobulin variable locus comprises VH, DH and JH gene segments, or Vκ and Jκ gene segments, or Vλ, and Jλ gene segments, or Vλ, Jλ and Cλ gene segments; b) providing a vector comprising an engineered partly canine immunoglobulin locus, said engineered partly canine immunoglobulin locus comprising chimeric canine immunoglobulin gene segments, wherein each of the partly canine immunoglobulin gene segment comprises canine immunoglobulin variable gene coding sequences and rodent non-coding regulatory or scaffold sequences, with the partly canine immunoglobulin variable gene locus being flanked by target sites for a site-specific recombinase wherein the target sites are capable of recombining with the target sites introduced into the rodent cell; c) introducing into the cell the vector and a site-specific recombinase capable of recognizing the target sites; d) allowing a recombination event to occur between the genome of the cell and the engineered partly canine immunoglobulin locus resulting in a replacement of the endogenous immunoglobulin variable gene locus with the engineered partly canine immunoglobulin locus; e) selecting a cell that comprises the engineered partly canine immunoglobulin variable locus generated in step d); and utilizing the cell to create a transgenic rodent comprising partly canine the engineered partly canine immunoglobulin variable locus. In some aspects, the cell is a rodent embryonic stem (ES) cell, and in some aspects the cell is a mouse embryonic stem (ES) cell. Some aspects of this method further comprise after, after step a) and before step b), a step of deleting the endogenous immunoglobulin variable gene locus by introduction of a recombinase that recognizes a first set of target sites, wherein the deleting step leaves in place at least one set of target sites that are not capable of recombining with one another in the rodent cell’s genome. In some aspects, the vector comprises canine VH, DH, and JH, coding sequences, and in some aspects the vector comprises canine VL and JL coding sequences. In some aspects, the vector further comprises a promoter, splice sites, and recombination signal sequences (col. 6, lines 23-67).
Wabl demonstrates successfully producing a transgenic mouse expressing canine immunoglobulins:
FIG. 10 is a schematic diagram illustrating the introduction of an engineered partly canine immunoglobulin λL chain variable region gene locus into the endogenous immunoglobulin λL chain locus of the mouse genome (col. 9, lines 38-41).
In a particularly preferred aspect, the transgenic non-canine rodent further comprises partly canine Igl loci comprising coding sequences of canine Vκ or Vλ, genes, and Jκ or Jλ genes, respectively, in conjunction with their intervening (non-coding regulatory or scaffold) sequences corresponding to the immunoglobulin intervening sequences present in the Ig1 loci of the rodent (col. 14, lines 46-52).
Example 5: Introduction of an Engineered Partly Canine Immunoglobulin Locus into the Immunoglobulin Lambda Chain Gene Locus of a Mouse Genome
Another method for replacing a portion of a mouse genome with an engineered partly canine immunoglobulin locus is illustrated in FIG. 10. This method comprises deleting approximately 194 Kb of DNA from the wild-type mouse immunoglobulin lambda locus (1001)—comprising Vλx/Vλ2 gene segments (1013), Jλ2/Cλ2 gene cluster (1015), and Vλ1 gene segment (1017)—by a homologous recombination process involving a targeting vector (1003) that shares identity with the locus both upstream of the Vλx/Vλ2 gene segments (1013) and downstream of the Vλ1 gene segment (1017) in the immediate vicinity of the J3, C3, J1 and C1 lambda gene cluster (1023). The vector replaces the 194 Kb of DNA with elements designed to permit a subsequent site-specific recombination in which a non-native piece of DNA is moved into the modified Vλ locus via RMCE (1004). In this example, the non-native DNA is a synthetic nucleic acid comprising both canine and mouse sequences.
The ES cell clones carrying the deletion in one of the two homologous copies of their immunoglobulin lambda chain locus are retransfected (1004) with a Cre recombinase expression vector together with a piece of DNA (1007) comprising a partly canine immunoglobulin lambda chain locus containing V, J and C region gene segments. The key features of this piece of DNA (1007) are as follows: a lox5171 site (1031); a neomycin resistance gene open reading frame lacking the initiator methionine codon, but in-frame and contiguous with an uninterrupted open reading frame in the lox5171 site (1047); a FRT site 1027); an array of 7 functional canine lambda variable region gene segments, each comprised of canine lambda coding sequences embedded in mouse lambda noncoding sequences (1051); an array of J-C units where each unit is comprised of a canine J2, gene segment and a mouse lambda constant domain gene segment embedded within noncoding sequences from the mouse lambda locus (1055) (the canine Jλ gene segments are those encoding Jλ1, Jλ2, Jλ6 and Jλ7, while the mouse lambda constant domain gene segments are C1 and/or C2 and/or C3); a mutated recognition site for the Flp recombinase known as an “F3” site (1043); an open reading frame conferring hygromycin resistance (1057), which is located on the antisense strand relative to the immunoglobulin gene segment coding information in the construct; a loxP site (1039) in opposite relative orientation to the lox5171 site.
The sequences of the canine Vλ and Jλ gene coding regions are in Table 3.
The ES cell clones carrying the partly canine immunoglobulin DNA (1011) in the mouse lambda chain locus are microinjected into mouse blastocysts from strain DBA/2 to create partially ES cell-derived chimeric mice according to standard procedures. Male chimeric mice with the highest levels of ES cell-derived contribution to their coats are selected for mating to female mice. The female mice of choice here are of the C57B1/6Ntac strain, which carry a transgene encoding the Flp recombinase expressed in their germline. Offspring from these matings are analyzed for the presence of the partly canine immunoglobulin lambda chain locus, and for loss of the FRT-flanked neomycin resistance gene and the F3-flanked hygromycin resistance gene that were created in the RMCE step. Mice that carry the partly canine locus are used to establish a colony of mice.
In some aspects, the mice comprising the canine-based heavy chain and kappa locus (as described in Examples 3 and 4) are bred to mice that carry the canine-based lambda locus. Mice generated from this type of breeding scheme are homozygous for the canine-based heavy chain locus, and can be homozygous for the K-K canine-based locus or the L-K canine-based locus. Alternatively, they can be heterozygous at the kappa locus carrying the K-K locus on one chromosome and the L-K locus on the other chromosome. Each of these mouse strains is homozygous for the canine-based lambda locus. Monoclonal antibodies recovered from these mice are comprised of canine heavy chain variable domains paired in some cases with canine kappa variable domains and in other cases with canine lambda variable domains. The lambda variable domains are derived from either the canine-based L-K locus or the canine-based lambda locus.
Wabl patented a transgenic mouse expressing canine VH, DH, JH, VL and JL coding sequences:
Claim 1. A transgenic mouse with a genome in which an entire endogenous immunoglobulin variable gene locus has been deleted and replaced with an engineered partly canine immunoglobulin locus comprising canine immunoglobulin variable gene VH, D and JH and/or canine VL and JL coding sequences and mouse immunoglobulin variable gene locus non-coding regulatory sequences, wherein the engineered partly canine immunoglobulin locus of the transgenic mouse is functional and expresses immunoglobulin chains comprised of canine variable domains and mouse constant domains.
Claim 7. A method for generating the transgenic mouse of claim 1, said method comprising:
a) integrating in a mouse embryonic stem (mES) cell’s genome at least one target site for a site-specific recombinase in a mES cell genome upstream of an endogenous immunoglobulin variable gene locus and at least one target site for a site-specific recombinase downstream of the endogenous immunoglobulin variable gene locus, wherein the endogenous immunoglobulin variable locus comprises VH, DH and JH gene segments, or Vκ and Jκ gene segments, or Vλ and Jλ segments, or Vλ, Jλ and Cλ gene segments,
b) providing a vector comprising an engineered partly canine immunoglobulin locus, said engineered partly canine immunoglobulin locus comprising partly canine immunoglobulin variable region segments, wherein each of the partly canine immunoglobulin variable region gene segments comprises canine immunoglobulin variable region gene VH, DH, and JH and/or canine VL and JL, coding sequences and mouse non-coding regulatory, with the partly canine immunoglobulin variable region gene locus being flanked by target sites for a site-specific recombinase, wherein the target sites are capable of recombining with the target sites introduced into the mES cell in step a),
c) introducing into the mES cell the vector of step b) and a site-specific recombinase capable of recognizing the target sites,
d) allowing a recombination event to occur between the genome of the mES cell and the engineered partly canine immunoglobulin locus, resulting in a replacement of the endogenous immunoglobulin variable gene locus with the engineered partly canine immunoglobulin locus,
e) selecting an mES cell that comprises the engineered partly canine immunoglobulin variable locus generated in step d); and
f) generating a transgenic mouse which expresses immunoglobulin chains comprised of canine variable domains and mouse constant domains with the mES cell of step e).
Wabl further teaches exemplary nucleotide sequences of canine immunoglobulin genes for producing transgenic mice including: heavy chain VH genes 1-77, DH genes 1-6, JH genes 1-5 in Table 1; light chain lambda genes VL 1-13, and JL 1-5 (Table 3).
Wabl teaches it is known the canine genome VH region comprises approximately 80 VH, 6 DH, and 3 JH gene segments mapping to a 1.28 Mb region of canine chromosome 8. The lambda coding region maps to canine chromosome 26, while the kappa coding region maps to canine chromosome 17 (col. 13, lines 30-41).
Wabl does not teach the transgenic mouse genome comprises specifically IGHV4-1, IGHV3-2, IGHV3-3, IGHV3-4, IGHD1-6, IGHJ1-6, IGLV3-1. IGLV3-2, IGLV3-3, IGLV3-4, IGLV4-5, IGLV4-6, IGLJ1-9, IGLC1-9, as well as IGHV3-5 genes.
Green, like Wabl, teaches and suggests a transgenic mouse comprising a genome comprising a nucleic acid encoding canine DNA, wherein the DNA encodes or expresses canine heavy chain VH, DH, and JH (also known as IGHV, IGHD, and IGHJ) gene segments, and Vλ (VL), Jλ (JL) and Cλ (CL) (also known as IGLV, IGLJ, AND IGLC) gene segments ([147]). Green teaches mice are able to generate antibody diversity ([50]).
Hwang teaches the canine immunoglobulin heavy chain repertoire and gene sequences of chromosome 8 were known and publicly available at CanFam 3.1 whole genome assembly (p. 182, col. 1, Methods).
NCBI genome assembly CanFam3.1 teaches the canine genome sequences are publicly available for chromosome 8 and 26.
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was effectively filed to include canine immunoglobulin VH, DH, JH , VL, JL, and CL genes IGHV4-1, IGHV3-2, IGHV3-3, IGHV3-4, IGHD1-6, IGHJ1-6, IGLV3-1. IGLV3-2, IGLV3-3, IGLV3-4, IGLV4-5, IGLV4-6, IGLJ1-9, IGLC1-9, and IGHV3-5 in the transgenic mouse of Wabl. One would have been motivated to, and have a reasonable expectation of success to because: (1) Wabl and Green teach and suggest replacing the endogenous Ig genes of a mouse with canine VH, DH, JH , VL, JL, and CL genes to produce a transgenic mouse expressing canine antibodies and demonstrate successfully producing transgenic mice expressing non-endogenous antibodies; (2) Wabl teaches the VH, DH, JH , VL, JL, and CL canine genes for producing the transgenic mouse are encoded on canine chromosome 8 and chromosome 26; (3) Hwang and NCBI genome assembly CanFam3.1 teach and demonstrate canine immunoglobulin gene sequences on chromosomes 8 and 26 were readily and publicly available.
Given: (1) Wabl and Green teach and suggest replacing the mouse Ig genes with canine VH, DH, JH , VL, JL, and CL genes to produce a transgenic mouse expressing canine antibodies, (2) Wabl teaches the sequences of canine VH, DH, JH , VL, JL, and CL genes are located on chromosomes 8 and 26; (3) Hwang and NCBI genome assembly CanFam3.1 demonstrate canine immunoglobulin gene sequences on chromosomes 8 and 26 were readily and publicly available; and (4) Wabl and Green demonstrate successfully replacing mouse Ig genes with non-endogenous Ig genes to produce a transgenic mouse; it is well within the level of the ordinary skilled artisan to replace the mouse Ig genes with any and all of the canine VH, DH, JH , VL, JL, and CL genes located on chromosome 8 and 26, encompassing instantly claimed IGHV4-1, IGHV3-2, IGHV3-3, IGHV3-4, IGHD1-6, IGHJ1-6, IGLV3-1. IGLV3-2, IGLV3-3, IGLV3-4, IGLV4-5, IGLV4-6, IGLJ1-9, IGLC1-9, and IGHV3-5 genes, in the transgenic rodent of Wabl with a reasonable expectation of success.
4. All other rejections recited in the Office Action mailed April 23, 2026 are hereby withdrawn in view of amendments.
5. Conclusion: No claim is allowed.
6. Any inquiry concerning this communication or earlier communications from the examiner should be directed to LAURA B GODDARD whose telephone number is (571)272-8788. The examiner can normally be reached Mon-Fri, 7am-3:30pm.
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/Laura B Goddard/Primary Examiner, Art Unit 1642
