WO1994011507A2 - Production of monoclonal recombinant antibodies without the use of hybridomas by in vitro spleen fragment culture combined with isothermal self-sustained sequence replication of rna - Google Patents
Production of monoclonal recombinant antibodies without the use of hybridomas by in vitro spleen fragment culture combined with isothermal self-sustained sequence replication of rna Download PDFInfo
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- WO1994011507A2 WO1994011507A2 PCT/US1993/011295 US9311295W WO9411507A2 WO 1994011507 A2 WO1994011507 A2 WO 1994011507A2 US 9311295 W US9311295 W US 9311295W WO 9411507 A2 WO9411507 A2 WO 9411507A2
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6865—Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
Definitions
- the present invention relates to methods for the
- the methods of the instant invention are based oh in vitro spleen fragment culture of B cells which produce
- the invention also relates to generic degenerate primer pools for use in replication of RNA encoding monoclonal antibodies of interest.
- Monoclonal antibodies are used in therapy, diagnostics, and basic research.
- monoclonal antibodies are directed against a toxin or a tumor surface antigen to mount the patient's immune response against the targeted toxin or pathogenic cell.
- a monoclonal antibody may be conjugated with a toxin, thereby directing the toxic action to the tumor cell.
- Ex vivo, or extracorporeal immuno-therapy involves the removal of the patient's bone marrow or blood and isolation of a selected cell population by means of binding a predetermined antigen on the cells' surface with a monoclonal antibody against that cell surface antigen.
- the monoclonal antibody may be
- Isolation of hemopoietic cells also offers the possibility of genetic manipulation of the patient's cells to correct a genetic defect or to expand a desired population of hemopoietic cells, which are thereafter returned to the patient. Immunodiagnostic tests are based on the generation of a signal
- hybridoma cells are the source of monoclonal antibodies which currently provides the most reproducible source of antibodies for immuno-therapy and immunodiagnostics.
- Production of hybridoma cell lines producing monoclonal antibodies of a desired specificity requires multiple experimental steps which may extend over a significant time period.
- the immunization, screening and hybridoma production stages all pose problematic challenges in which costs increase in proportion to the amount of time and effort needed. Additional difficulties are at times encountered even after a monoclonal hybridoma cell line is identified and clonally selected.
- An expanded population of hybridoma cells can lose its ability to produce a specific monoclonal antibody after prolonged growth in culture. This propensity to lose antibody expression is also observed in hybridoma cell lines that have been frozen and stored after clonal selection.
- hybridoma cell line producing a monoclonal antibody of interest has frequently proven to be problematical.
- This RT/PCR failure has been attributed to the complication of non-productive immunoglobulin rearrangements which increase the complexity of the amplification-generated cDNA library. In turn, this significantly increases the likelihood of missing clones containing the productive immunoglobulin rearrangements.
- Fusions of the phage pVIII protein also have been used to display antibody fragments on the surface of the filamentous phage (Chang, CN., et al., (1991) J Immunology 147:3610-3614; Huse, W.E., (1991) In: Borrebaeck, D.A.K. (ed.), Antibody Engineering.
- Binding of the displayed antibody on the surface of the phage with antigen can be used as a way to recover antibodies composed of the original or novel V H and V L gene pairings. However, because each phage contains only one such V H and V L pairing, the frequency of the
- the size of a phage display library may have to be even greater than 10 11 copies.
- T and secondary B cells which increase in specificity for the antigen and enable the organism to mount a more vigorous response upon additional exposure to the same antigen.
- a primary antibody is composed of a light chain and a heavy chain, which are connected by a
- this complex is associated with other light/heavy chain complexes in either dimeric or pentameric forms.
- Secondary B cells differ from primary B cells with respect to the variable regions which dominate a
- variable gene cassettes from which a single variable gene is selected in response to an antigen.
- Cells which carry an appropriate gene cassette are known as "responsive B cells" for that particular antigen, even if they have not been previously exposed to that particular antigen, i.e. the responsive B cells have been pre-programmed to respond to the general three-dimensional configuration and physico-chemical attributes of the antigen of interest.
- Secondary B cells differ from primary B cells in their propensity to accumulate somatic mutations in the variable region to produce antibodies with substantially higher affinities for the antigen than those produced by primary B cells.
- the resulting amplified DNA would be expected to contain a complex mixture of the gene of interest and other genes. This situation would be expected to contain a complex mixture of the gene of interest and other genes. This situation would be expected to contain a complex mixture of the gene of interest and other genes. This situation would be possible to contain a complex mixture of the gene of interest and other genes. This situation would be possible to contain a complex mixture of the gene of interest and other genes. This situation would be possible to contain a complex mixture of the gene of interest and other genes. This situation would
- nucleotide sequences encoding only the desired H and L chains.
- the invention is directed to methods for obtaining an amplified RNA encoding a predetermined chain of a desired antibody against a specific antigen. Briefly, the methods involve the steps of
- the methods of the invention can be extended to produce a desired recombinant monoclonal antibody by converting RNA encoding the heavy chain and RNA encoding the light chain to cDNA.
- the cDNA is converted into double-stranded DNA product by primer extension or RT/PCR and is then
- the host cells incorporated into one or two expression vectors, which are transfected into host cells.
- the host cells are incorporated into one or two expression vectors, which are transfected into host cells.
- the invention is also directed to a method for
- the invention is also directed to degenerate primer pools generic for mouse immunoglobulin light and heavy chains.
- B cell encompasses the entire category of cells which lead to antibody production.
- B cell refers to a mammalian cell of hemopoietic origin whose progeny produce antibodies.
- B cell also includes stem cells or precursor cells which have the capacity to proliferate to produce progeny which
- B cell also includes “memory B cells” or “secondary B cells” that have been generated as the result of a previous immunization.
- antigenic molecule refers to a molecule which elicits an immune response in the form of antibodies produced by B cells.
- the antigenic molecule may be a protein or other molecule of biological or environmental origin.
- antibodies may be produced against a protein, a nucleic acid, or a steroidal hormone when B cells are stimulated by the specific molecule.
- responsive B cell refers to a B cell which contains an immunoglobulin gene rearrangement which enables the B cell to produce an antibody against a specific antigen.
- a responsive B cell may be a precursor cell which contains a gene cassette encoding an antibody of low specificity for the general three dimensional configuration and physico-chemical properties of the specific antigen. Later in differentiation, a responsive B cell may contain an immunoglobulin gene rearrangement which encodes an antibody of greater specificity and affinity for the specific antigen.
- B cell is understood to include "responsive B cells”.
- antibody minimally refers to an immunoglobulin having at least one light and one heavy chain (Fab) , or at least two light chains and two heavy chains, each chain having a variable and possibly a constant region.
- the antibody may be an IgG, an IgA, an IgE, an IgM, or an IgD.
- monoclonal antibodies refers to antibodies which are identical to each other because they have been produced by cells arising from a single cell clone expressing defined H and L chain genes encoding the antibodies.
- recombinant antibody refers to an antibody encoded on an expression vector contained in host cells and expressed as a protein by the host cells.
- recombinant antibody may be in the form of heavy chains and light chains bound by di-sulfide bonds, just as a naturally occurring antibody.
- a naturally occurring antibody Alternatively, a
- recombinant antibody may be a single-chain antibody construct which has the capability to bind a specific antigen.
- heterogeneous RNA refers to RNA which encodes many different cellular proteins.
- heterogeneous RNA encompasses total RNA extracted from a culture of cells, as well as poly(A) + RNA isolated from total RNA extract.
- target RNA refers to RNA which encodes a single protein.
- 3SR refers to isothermal self-sustained sequence replication.
- isothermal self- sustained sequence replication refers to a process by which a target RNA is amplified in a process which essentially mimics the way in which retroviral RNA is amplified in nature.
- Alternative terms are 3SR and TAS.
- the process makes use of three different enzyme activities: reverse transcriptase, RNA polymerase, and RNaseH.
- isothermal refers to the fact that the process is carried out at a single or narrow
- genetic degenerate primer pool refers to a degenerate primer pool in which the variations are limited to those sequence substitutions represented in a catalogue of sequences known for a specified
- a degenerate primer pool generic for mouse IgG light chain would contain sequences encoding a 3' constant region as well as all possible sequences known for 5' variable regions of all known mouse IgG families such as lambda and kappa.
- Figure 1 depicts the major steps in producing a
- Figure 2 defines the sequences of oligonucleotide primers referred to herein by the number in the far left column of Figure 2.
- Figure 3 shows Northern (A) and Southern (B) blot
- HC heavy chain
- M size markers of Mspl digested pBR322 fragments
- S anti -PC specific 5'end primers
- G v generic variah-te region 5'end primers
- G L generic leader region 5'end primers
- Figure 3 is a scheme for the construction of plasmid pT15-110 which encodes a single chain (sc) antibody form of the anti-PC antibody.
- MBS metal binding site
- TAG c- myc tag
- LC light chain
- HC heavy chain.
- Figure 5 shows nuolectide and amino aoid sequence of the four heavy (4A) and light (4B) chain groups (G 1-4 Cloned from spleen fragment culture D 1 A 6 (anti-E2).
- the CDR regions are noted in the heavy and light chain groups by overlining the appropriate sequences.
- the heavy chain sequences include the 5'end variable region primers ( Figure 2) and the light chain sequences include only the primers three nucleotides of the 5'end variable region primers ( Figure 2).
- B cells are isolated from a donor mammal that has been previously immunized with a specific antigen.
- B cells may be further selected for precursor status by their lack of memory B cell markers.
- Isolated B cells are injected into a recipient mammal without viable endogenous immune system cells.
- the recipient may have been previously immunized with all or part of the antigen to promote T cell helper function.
- the injected B cells are allowed to colonize the recipient's spleen.
- Spleen of the recipient is removed and dissected, each fragment being placed in a separate culture well.
- Spleen fragments are maintained in culture and stimulated with antigen. Certain fragments contain responsive B cells which will proliferate to produce progeny which synthisize antibody against the antigen. Each culture is assayed for an antibody of interest, based on desired affinity and specificity for the antigen. A culture containing a positive fragment is identified. The positive fragment is extracted for total RNA.
- RNA from the positive fragment is subjected to 3SR amplification of the RNA encoding the antibody of interest.
- Generic degenerate primer pools are provided herein for the amplification of RNA encoding mouse immunoglobulin light and heavy chains.
- RNA encoding the antibody of interest is converted into a double-stranded cDNA.
- Double stranded cDNA encoding the antibody of interest is incorporated into an expression vector.
- the expression vector is transfected into a host cell.
- the desired antibody is expressed by the host cell and isolated.
- a donor animal such as a mouse is immunized with an antigen of interest such as estradiol. Injection of antigen will stimulate the proliferation and
- B cells which produce a primary antibody against the antigen as well as memory B cells.
- a naive animal i.e. one which has not been injected with antigen, may be a donor of B cells since it is expected that certain precursor B cells possess gene rearrangements encoding antibodies of low specificity for a large repertoire of antigenic
- B cells are isolated from the donor animal's bone marrow, peripheral blood, or spleen.
- B cells may be isolated immediately from a naive donor.
- the isolated B cells may be subjected to a further selection process whereby cells which exhibit low binding capacity for J11D monoclonal antibody [J11D lo cells] are selected for injection (Litton, et al., Cell 59:1049-1059, 1989).
- J11D lo cell population is considered to be enriched for secondary B cells, which are expected to produce antibodies of higher affinity and specificity for the antigen of interest.
- Isolated B cells are injected into a recipient animal which does not possess viable endogenous immune system cells.
- the recipient animal may have been "lethally irradiated” such that essentially all its immune system cells have been destroyed.
- the term "lethally irradiated" such that essentially all its immune system cells have been destroyed.
- irradiated refers to the killing of immune system cells and does not refer to killing of the animal itself.
- the recipient animal for instance an SCID mouse, may be genetically deprived of an immune system.
- the spleen of the recipient animal is denuded of endogenous B cells and is open for colonization by injected B cells.
- the concentration of injected B cells is calculated so that, on average, only one responsive B cell will
- the recipient animal is also immunized with antigen to stimulate helper T-cell function, which facilitates B cell proliferation and differentiation. If an immune deficient recipient is used, the transfer of antigen stimulated helper T cells is needed to facilitate B cell proliferation and differentiation.
- the spleen is then removed from the recipient animal and dissected into fragments, the size of the fragments being chosen so that each fragment contains, on the average, only one responsive B cell.
- the size of the fragments being chosen so that each fragment contains, on the average, only one responsive B cell.
- fragment size is about 1 mm 3 .
- Each spleen fragment is placed in a separate culture well, typically in 96 well plates.
- the fragments are maintained in culture as described in Example 1 below.
- an aliquot of the antigen of interest is added to each culture well.
- the antigen of interest is added to each culture well.
- the medium of each culture well is assayed for affinity and specificity for the antigen of interest.
- a positive well which contains a spleen fragment having B cells producing the antibody of interest.
- the spleen fragment technique for obtaining monoclonal antibodies has several advantages over hybridoma technology. The time and expense involved in production and screening of spleen fragments is expected to be less than the time and expense involved in
- the spectrum of antibody products may be much greater using the spleen fragment technique.
- RNA is extracted from the identified spleen fragment.
- poly(A) + RNA may be isolated from the total RNA. In either case, the resulting
- RNA heterogeneous RNA encodes many proteins synthesized by the B cells.
- the target RNA species which encode light and heavy chains for the antibody of interest, are expected to represent only a portion of the total RNA or the poly (A) + RNA extracted therefrom.
- the next task is to amplify the RNA which encodes the heavy and light chains of the antibody of interest. This task is made difficult by the inevitable presence of non- desired extraneous nucleotide sequences which have certain homologies to the RNA of interest but which, if amplified, will yield a non-productive genetic sequence. That is, amplified extraneous nucleotides would probably not encode a functioning antibody, and they certainly would not encode the previously identified antibody of interest.
- PCR polymerase chain reaction
- RNA transcription and RNA transcription steps replicates a nucleic acid target by means of cDNA intermediates.
- Two or three different enzymes work together in this system; typically a reverse transcriptase, an RNaseH, and an RNA polymerase are required.
- exogenous RNaseH i.e. from E. coli
- 3SR can be conducted in a single pot at a single temperature, below 95°C, which allows the activity of the enzymes in the reaction. The temperature is chosen to optimize the activity of the specific enzymes.
- 3SR enzymes are optimally active in the temperature range of 37°C to 42°C.
- new enzymes are in development which may be optimally active at substantially higher temperatures, although the 3SR reaction will routinely be conducted below about 95°C.
- the RNA of interest is targeted by a primer set including a specific 3' primer and a 5' primer pool.
- the 3'primer encodes a T7 promoter as well as antisense for the RNA of interest. Since the constant region of each type of immunoglobulin generally has a well defined single sequence, the 3' primer can be clearly defined. Primer sequences for the 3' region of mouse immunoglobulin heavy and light chains are listed in Figure 2 (91-267*, 91- 268*, 920-004, 92-006, 92-002*).
- variable chain family is present in the antibody of interest. Therefore, a degenerate 5' primer pool, generic for all variable chain families of the animal species of interest, is designed to enable
- Degenerate primers were designed using the data base of Kabat et al. (In: Sequences of Proteins of Immunological Interest, 4th Edition (1987) U.S. Dept. Health and Human Services), taking into account codon degeneracies for each amino acid in the conserved sequences of the leader and FR1 regions.
- the degenerate pool generic for the light chain is depicted in Figure 2 (sequences 92-099 to 92-102), for the heavy chain starting with the leader sequence in Figure 2 (sequences 92-107 and 92-108), and for the heavy chain starting with the coding region for the mature protein in Figure 2 (sequences 92-095 to 92- 098 and 92-109 to 92-110).
- Oligonucleotide primers were synthesized using an Applied Biosystems Incorporated DNA synthesizer using
- sub-pool 92-099 was begun by the addition starting at the 3'end of A, then C, followed by C. Then A was specifically added at the degenerate site four nucleotides from the 3' end.
- Oligonucleotide synthesis was continued with the addition of C followed by T. Then A and G were added to the reaction mix in 1:1 proportion for addition at the heterogeneous site 7 nucleotides from the 3 'end.
- reaction mix in the proportion of 1:1:1 for addition at the heterogeneous site 10 nucleotides from the end.
- the degenerate primer pool generic for the mouse immunoglobulin light chain comprises a 5' primer pool having an equal proportion of I, II, III, and IV, wherein
- the degenerate primer pool for the mouse IgG light chain further comprises a 3' primer having the sequence 92-002* or 92-006 in Figure 2, said degenerate primer pool comprising one part of said 3' primer and four parts of said 5' primer pool.
- This proportion of 1:4, 3' pool: 5' pool facilitates the efficient amplification of the RNA of interest because it increases the chances that the appropriate 5' sequence will be present in proportion to the 3' sequence.
- immunoglobulin heavy chain coding from the leader sequence, may be constructed to contain an equal
- X 1 G or A
- X 2 G or C
- X 3 G or C
- X 4 T or G
- X 5 T or C
- X 6 A or C
- X 7 A or G
- X 8 G or T
- X 9 G or A
- X 10 G or A
- X n G or C
- X 12 G or C
- X 13 C or
- X 14 T or A
- X 15 T or C.
- the above 5' primer pool for the heavy chain leader sequence is combined with a 3' primer specific for mouse IgG heavy chain constant region.
- the 3' primer has the sequence 91-267*, 91-268*, or 92-004 in Figure 2.
- the total primer pool comprises one part of the 3' primer and two parts of the 5' primer pool.
- immunoglobulin heavy chain coding from the mature amino acid sequence, may be constructed to contain an equal proportion of I, II, III, and IV, wherein
- X 1 G, C, or A
- X 2 G, C, or A
- X 3 A or G.
- the above 5' primer pool for the heavy chain mature sequence is combined with a 3' primer specific for mouse IgG heavy chain constant region.
- the 3' primer has the sequence 91-267*, 91-268*, or 92-004 in Figure 2.
- the total primer pool comprises one part of the 3' primer and four parts of the 3' primer pool.
- target RNA's encoding the light chain and the heavy chain are
- Q Beta Replication is based on a phage enzyme which replicates negative strand RNA into positive strand RNA, thus also providing preferential amplification of RNA.
- complementary double stranded DNA is made using primer extension or RT/PCR. Once the cDNA for each chain of the antibody of interest is obtained, there are several routes to choose for making the recombinant antibody.
- Each double stranded DNA of the 3SR products can be inserted into a separate expression vector, both vectors can be transfected into a host cell such as E. coli, and the host cell will synthesize both immunoglobulin chains and cause them to associate properly via di-sulfide bonds to form a functioning antibody.
- a plasmid containing both light and heavy chain genes under the regulation of separate promotors may achieve expression of each of the antibody chains.
- the two Ig chains can then be expressed by a single host cell.
- each vector can be transfected into a separate host cell, each of which produces a light or a heavy chain
- the two immunoglobulin chains can then be isolated from the host cells and caused to form
- the invention makes possible the preselection for: 1) specific isotypes, 2) antibodies, with binding affinities of desired levels, and 3) defined specificity as determined by competitive analog screening. Using the methods of the invention, screening is performed on a relatively small library of antibodies which were
- nucleotide sequences encoding predominantly the desired antibody chains are selectively amplified, cloned, and expressed.
- T15 refers to an epitope of certain antibodies specific for the Pneumococccus coat protein (PC) which is found on two hybridoma cell lines (R2-26 and R2-09) that produce anti-PC monoclonal antibodies. Nucleotide sequences for both light and heavy chains of T15 antibodies have been determined previously. The T15-specific primers were selected from published sequences for the V k T15(S107) light chain rearrangement and V H S107 T15 heavy chain. The sequences of the specific primer pair are listed in
- Pegenerate primers were designed such that members of each set would hybridize to representatives of several gene families. This approach was undertaken because antibodies of future interest may belong to any of several gene families.
- V H -leader heavy chain leader
- V H heavy chain gene variable region
- V L light chain gene variable region
- the 3'-end constant region of the light (C L ) and heavy chain (C H 1) genes have relatively little sequence variation.
- the most common type of antibody light chain is k and the predominant isotype for heavy chain from secondary cells is IgG.
- the 3' end primers used for the amplification of the kappa light chain and IgGl heavy chain of anti-PC with T15-specific primers in Section V.C.I were used in combination with the 5'-end degenerate primers.
- degenerate primers were designed to contain a Sail restriction enzyme site at their 5' ends to aid in the cloning of the genes; however, no restriction enzyme sites are present in the degenerate V H -leader primers.
- Pegenerate primers were designed using the data base of Kabat et al. (In: Sequences of Proteins of Immunological Interest, 4th Edition (1987) U.S. Pept. Health and Human Services), taking into account codon degeneracies for each amino acid in the conserved sequences of the leader and FR1 regions. The rationale for the design of this degenerate pool is described above in the Pescription section.
- the degenerate pool generic for the light chain is depicted in Figure 2 (92-099 to 92-102), for the heavy chain starting with the leader sequence in Figure 2 (92- 107 to 92-108), and for the heavy chain starting with coding region for the mature protein in Figure 2 (92-095 to 92-098 and 92-109 to 92-110).
- Figure 2 sequences 92-099, 92-100, 92-101, and 92-102] illustrates the principle for synthesis of degenerate pools. Briefly, the oligonucleotide synthesis of sub- pool 92-099 was begun by the addition starting at the 3'end of A, then C, followed by C. Then A was
- Oligonucleotide synthesis was continued with the addition of C followed by T. Then A and G were added to the reaction mix in 1:1 proportion for addition at the heterogeneous site 7 nucleotides from the 3'end. Synthesis was continued with the specific addition of A followed by C. Then G,C, and A were added to the reaction mix in the proportion of 1:1:1 for addition at the heterogeneous site 10 nucleotides from the end. Oligonucleotide synthesis was continued in this manner until degenerate sub-pool 92-099 was complete. Each degenerate sub-pool was synthesized in this manner, resulting in four separate primer sub-pools. Then the sub-pools were combined in equal proportion to form the light chain 5'degenerate primer pool generic for mouse light chain immunoglobulin. By this method, it was assured that the total pool would have an equal
- the final concentration of each of the T15-specific primers in the 3SR, cPNA, and PCR reactions used in the successful isolation of heavy and light chain genes was 0.1 ⁇ M, 1.0 ⁇ M, and 0.2 ⁇ M, respectively.
- the 3SR reaction was modified for the degenerate primers as follows: the concentration of each primer in the
- reaction was either 0.025 ⁇ M or 0.25 ⁇ M, and the reaction was allowed to proceed for 1.5 hours.
- the PCR reaction was modified by increasing the concentration of the 5'- end degenerate primers for the cPNA reaction as described below.
- Heavy chain, leader region Aliquots of approximately 0.9 ⁇ g of total RNA from PC-specific hybridoma line R2-09 were amplified by 3SR with primers 92-(107, 108)/91- 268* ( Figure 2). Sequences for the degenerate primers 92-
- Heavy chain, variable region Aliquots of approximately 0.9 ⁇ g of total RNA from PC-specific hybridoma line R2-09 were amplified by 3SR with primers 92- (95-98) /91- 268* ( Figure 2). Sequences for degenerate primers 92-(95-
- Light chain variable region Aliquots of approximately 0.9 ⁇ g of total RNA from PC-specific hybridoma line R2-09 were amplified by 3SR with primers 92-(99-102)/92- 002 ( Figure 2). Sequences for the degenerate primers 92- (99-102) hybridize to the 5' end of the variable region of multiple light chain gene families and contain a Sail restriction enzyme site at their 5' ends. Primer 92-002 hybridizes to the 5' end of the light chain constant region (C L )
- 3SR products were detected by immobilizing three serial dilutions of each reaction on a nylon filter (Bio-Rad Zeta-Probe ® ) utilizing a slot blot apparatus (Schleicher and Schuell). Aliquots corresponding to 2 ⁇ l, 0.2 ⁇ l and 0.02 ⁇ l of the reaction were immobolized on the blot. Each blot included 3SR amplifications of HIV-1 as a positive control. The blots were probed with 32P-labeled oligonucleotides specific for the RNA of interest.
- oligonucleotides 92-008 and 92-009 ( Figure 2), which are anti-sense and sense probes, respectively, homologous for a relatively conserved sequence in the J-C H 1 region, specifically J H 1, J H 2, and J H 4.
- Primers 92-012 and 92-013 hybridize to the 5' end of the constant region of the light chain.
- Northern analyses Northern blot analyses were performed on a NuPAGE 8% RNA Gel Kit (Novex) according to the manufacturer's instructions. Aliquots of 3SR
- amplification products were denatured by diluting each sample 1:1 in 2xNuPAGE Urea Sample Buffer (Novex) and heating for 2 min at 85°C before separation. Following electrophoresis, transfer of the nucleic acids onto a nylon filter (Bio-Rad Zeta-Probe ® ) was carried out for 45 min at 0.4 amps in 1xNuPAGE Running Buffer (Novex) on a transfer apparatus (Hoefer, TE22). The PNA was cross- linked to the filter by UV irradiation at 0.125
- Each blot included a 3SR amplification of HIV-l as a positive control .
- the blots were probed with
- RT/PCR essentially according to the manufacturer's specifications (Perkin Elmer Cetus GeneAmp ® RNA PCR Kit).
- the thermal cycle parameters with T-15 specific primers were: 1 cycle for 2 min at 94°C; 3 cycles of 1 min at 94°C for denaturation, 1.5 min at 42°C for primer
- parameters for the degenerate primers were: 2 cycles of 1 min at 94°C for denaturation, 1 min at 42°C for primer annealing, and 2 min at 72°C for elongation; 30 cycles of 1 min at 94°C, 1 min 60°C, and 2 min at 72°C; followed by 10 min at 72°C and a 4° soak.
- the blots were probed with 32P-labeled oligonucleotides specific for the gene of interest.
- the blots included an Mspl digest of pBR322 PNA (New England Biolabs) that had been 32 P- labeled as molecular weight markers. Results :
- T15-specific primers In slot blot and Northern analyses of the 3SR amplification products, heavy chain variable region products were detected in amplifications of R2-09 total RNA, and light chain variable region products were detected in amplifications from R2-26 poly(A)+ RNA.
- the first PCR reaction employed primer pairs 92-194 and 92- 188, and 92-195 and 92-201 (Table 1) to amplify the heavy and light chain inserts, respectively ( Figure 4).
- Primers 92-188 and 92-201 contain overlapping linker sequences based on the Genex 212 linker (Bird, R.E., et al., 1988 Science 242:423-426).
- the PCR fragments produced by the first PCR step were then joined by a second PCR reaction with the primer pair 92-194 and 92- 195 ( Figure 4, Table 1).
- the resulting PCR fragment contained Sall and Xmal cloning sites at the termini (figure 4).
- V H Heavy chain variable region
- V L light chain variable region
- Plasmid pT15-110 was constructed for the expression of anti-PC sc antibody in E. coli.
- the gene for PC sc antibody is under the regulation of the alkaline phosphatase (phoA) promoter region and the Bacillus thuringiensis cry transcription terminator (Wong, H.C., et al., 1986 PNAS 83:3233-3237).
- the phoA leader is under the regulation of the alkaline phosphatase (phoA) promoter region and the Bacillus thuringiensis cry transcription terminator (Wong, H.C., et al., 1986 PNAS 83:3233-3237).
- nucleotide sequence is used to direct secretion of the antibody. Additionally, in pT15-100, a nucleotide sequence encoding five histidine residues is fused to the 3' end of the PC sc antibody gene. These histidine residues function as a metal binding site (MBS) and can be used in a rapid partial purification of the antibody.
- MFS metal binding site
- a nucleotide sequence encoding the 13 amino acids from the carboxy terminus of the human c-myc protein (Ramsey, G., et al., 1985 Mol. Cell Biol. 5:3610-3616) is also fused to the 5' end of the antibody gene. These amino acids provide an immunological tag useful for monitoring the expression and purification in Western blots and functionality in ELISA assays.
- Expression vector P15-110 was constructed as follows ( Figure 4). The phoA promoter, and leader sequence and the cry terminator were obtained in plasmid pSYC 1087. Oligonucleotide 92-183
- promoter in pSYC 1087 was removed by digestion of pMBS-4 with Clal and Xbal. The ends were repaired by Klenow PNA polymerase and religated to form plasmid pMBS-101.
- oligonucleotide 93-011 To form pPHO 101, oligonucleotide 93-011
- E. coli strain MM294 was transformed with pT15-110 and used as a host for the expression of anti-PC sc antibody.
- Cells transformed with plasmid pPHO-101 served as a negative control.
- Individual E. coli transformants were grown at 30°C to approximately 4-5 OP 600 in phosphate medium (1x MOPS, 0.4% glucose, 0.15% vitamin-free
- E. coli cultures were grown under low phosphate conditions for 7 hours to achieve maximal anti-PC sc antibody expression.
- Cell lysis and antibody purification Cell lysis was accomplished by resuspending transformed cells (20 OP 600 ) in 1.0 ml of sonication buffer (50 mM Na phosphate, pH 8.0, 300 mM NaCl, 0.25% Tween-20, 0.1 mM EGTA, 1 mM phenylmethylsulfonylchloride). The cells were frozen on dry ice/ethanol, thawed and sonicated on ice (10 cycles of 10 second bursts with 1 min cooling at 20 watts with a Bronson sonifier, [Panbury, CT], Model 450). The lysed cells were centrifuged at 11,000 rpm for 20 min at 4°C. The supernatant represented the total soluble protein. The total protein concentration in the supernatant was measured by the Lowry method with a PC Protein Asssay Kit (BioRad, Richmond, CA).
- sonication buffer 50 mM Na phosphate, pH 8.0, 300 mM
- Anti-PC sc antibody was partially purified from cell lysates using a Nickel (Ni)-NTA resin (Qiagen, Chatsworth, CA) according to the manufacturer's recommendations. Briefly, a 50% slurry of Ni-NTA resin (previously epuilibrated in sonication buffer) was added to an aliquot of supernatant cell lysate and agitated for 1 hour at 4°C. The resin was centrifuged at 14,000 rpm and the unbound fraction was collected. The resin was then washed with 1.0 ml aliquots of 10 to 100 mM imidazole dissolved in
- sonication buffer was applied to the resin in 1.0 ml aliquots. Aliquots were analyzed by Western blot using monoclonal antisera specific to c-myc (Oncogene Sciences, Uniondale, NY). ELISA assays were performed by coating each well of a 96 well microtiter plate with 50 ⁇ l of 50 ⁇ g/ml of phosphorylcholine conjugated to bovine serum albumin (PC-BSA) in 0.05 M carbonate buffer. Aliquots of E. coli lysates or fractions eluted from the Ni-NTA resin were added and reacted at room temperature for 4 hours.
- PC-BSA bovine serum albumin
- the secondary antibody reaction in which the mouse anti- c-myc binds to the anti-PC sc antibody, was allowed to incubate 4 hours at room temperature.
- the bound anti-c- myc antibody was visualized by incubation with alkaline phosphatase-labeled goat anti-mouse Ig antibody followed by reaction with p-nitrophenyl-phosphate (NPP). After color development, the plates were read at 405 nm on a Dynatech (Chantilly, VA) MR5000 microtiter plate reader.
- the fraction eluted with 40 mM imidazole from the Ni-NTA resin was the only material that demontrated quantitative binding to the immobilized phosphorylcholine. Because the undenatured anti-PC sc antibody that was present in the unbound fraction from the Ni-NTA resin did not react in the ELISA assay, it may be condluded that the majority of the anti- PC sc antibody made in E. coli is folded such taht it does not recognize the immobilized phosphorylcholine and/or the c-myc tag is also unavailable for the second antibody. Such improperly folded sc antibody can be denatured and refolded into a functional form as previously demonstrated for an anti-PC sc antibody
- the amount of the sc anti-PC antibody produced by pT15- 110 in E. coli was estimated using silver-stained SDS- PAGE to be approximately 0.1% of the total cell protein.
- Ponor BALB/c mice were each injected twice with 100 ⁇ g estradiol b coupled to Limulus polyphemus hemocyanin (Hy) (gift from F. Boches) at two month intervals, as
- This total nucleic acid was used for 3SR amplification and subsequent cloning of Ig light and heavy chain cPNAs.
- each 5'- and 3'-end primer concentration of each 5'- and 3'-end primer was 0.25 and 1.0 mM, respectively.
- the conditions used for the thermal cycling were: 30 cycles, each cycle consisting of
- the gel was soaked for 10 minutes in 0.05 M NaOH and 5 minutes in 1x TBE.
- Transfer of the nucleic acids onto Zeta Probe ® nylon filters was carried out for 45 minutes at 0.4 amps in 1x TBE buffer on a TE22 transfer apparatus. After the transfer, the nylon membrane was washed for 5 minutes each in 0.1 M NaOH and H 2 O.
- the PNA was cross-linked to the filter by UV irradiation at 0.15 joules/cm.
- the filters were probed with P-labeled oligonucleotides 92-009 or 92-013 ( Figure 2).
- the anti-E2 heavy chain variable region amplification reaction (10 ⁇ l) was digested with Sail and EcoRI because both restriction endonuclease sites were engineered into primers 92- (109-110) and 92- 140, respectively, for cloning purposes.
- the digested PCR fragments were gel purified and ligated into pUC18 which had been previously digested with Sail and EcoRI and treated with CAP.
- amplification reaction (10 ⁇ l) was treated with Klenow PNA polymerase and digested with Sail.
- the PCR fragments were gel purified and ligated into Smal and Sail digested pUC18 or pBR322 which had been treated with CAP.
- nucleotide sequence representative of clones from each allelic group is presented in Figure 5.
- the heavy chain clones from groups 2, 3 and 4 exhibited 81-86% similarity with the J558 V H heavy chain family (Kabat, et al., supra; Brodeur, P.H., et al., J Exp Med 168:2261-2278), whereas clones from group 1 were marginally similar to the S107 (79.7%) V H family.
- comparison of the sequences of clones of group 1 to sequences in GENBANK revealed a 96% similarity to a mouse anti-PNA rearranged heavy chain variable region (Kofler, R., et al., J Clin Invest
- Light chain clones used the J ⁇ 5 minigene while clones from group 1 used the J ⁇ 4 minigene.
- the leader region primers allowed for unambiguous determination of the sequences present at the amino terminus of the V H minigene.
- group 1 clones derived from the leader region primers, the nucleotide sequences predicted amino acids Val-5, Glu-6 and Thr-7 in place of Gln-5, Gln-6, and Ser-7 as predicted by using the generic variable region primers.
- Heavy and Light Chain Clones Obtained from Spleen Fragment Cultiure D 1 A 6 group 4 clones derived from the leader primers contained the codon for the amino acid Leu at position 3 in place of Lys as predicted by the variable region primers.
- the clones sequenced in each of the four groups of light and heavy chain variable regions were productive, indicating that more than one memory B cell colonized the splenic fragment D 1 A 6 .
- limiting dilution of transferred lymphocytes can be performed to obtain only one colonizing antigen responsive B cell.
- RNA encoding heavy and light chains of anti-estradiol, as produced in Example 4 is converted to cPNA.
- the cPNA when converted to double stranded PNA, is incorporated into expression vectors, which are then transfected into E. coli host cells.
- the host cells are induced to express the heavy chain and the light chain recombinant proteins for the anti-estradiol antibody.
- the heavy and light chains associate and form the appropriate disulfide bridges to form the complete anti-estradiol antibody.
- the antibody is isolated and purified from the host cell debris.
- the resulting recombinant monoclonal antibody possesses the desired specificity and affinity for estradiol as identified in the asssays of the
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Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
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EP94902330A EP0628076A1 (en) | 1992-11-19 | 1993-11-19 | PRODUCTION OF MONOCLONAL RECOMBINANT ANTIBODIES WITHOUT THE USE OF HYBRIDOMAS BY $i(IN VITRO) SPLEEN FRAGMENT CULTURE COMBINED WITH ISOTHERMAL SELF-SUSTAINED SEQUENCE REPLICATION OF RNA |
AU56737/94A AU5673794A (en) | 1992-11-19 | 1993-11-19 | Production of monoclonal recombinant antibodies without the use of hybridomas by (in vitro) spleen fragment culture combined with isothermal self-sustained sequence replication of rna |
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US97883592A | 1992-11-19 | 1992-11-19 | |
US07/978,835 | 1992-11-19 |
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WO1994011507A2 true WO1994011507A2 (en) | 1994-05-26 |
WO1994011507A3 WO1994011507A3 (en) | 1994-07-07 |
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PCT/US1993/011295 WO1994011507A2 (en) | 1992-11-19 | 1993-11-19 | Production of monoclonal recombinant antibodies without the use of hybridomas by in vitro spleen fragment culture combined with isothermal self-sustained sequence replication of rna |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000020460A1 (en) * | 1998-10-05 | 2000-04-13 | Ludwig Institute For Cancer Research | Methods for producing human tumor antigen specific antibodies |
EP1392866A2 (en) * | 2001-05-14 | 2004-03-03 | Henry Hongjun Ji | Novel method for cloning variable domain sequences of immunological gene repertoire |
WO2012092376A3 (en) * | 2010-12-31 | 2012-12-06 | Short Jay M | Comprehensive monoclonal antibody generation |
US10563254B2 (en) | 2007-01-23 | 2020-02-18 | Cambridge Enterprise Limited | Nucleic acid amplification and testing |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1992008800A1 (en) * | 1990-11-13 | 1992-05-29 | Siska Diagnostics, Inc. | Nucleic acid amplification by two-enzyme, self-sustained sequence replication |
-
1993
- 1993-11-19 WO PCT/US1993/011295 patent/WO1994011507A2/en not_active Application Discontinuation
- 1993-11-19 EP EP94902330A patent/EP0628076A1/en not_active Withdrawn
- 1993-11-19 AU AU56737/94A patent/AU5673794A/en not_active Abandoned
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1992008800A1 (en) * | 1990-11-13 | 1992-05-29 | Siska Diagnostics, Inc. | Nucleic acid amplification by two-enzyme, self-sustained sequence replication |
Non-Patent Citations (1)
Title |
---|
CELL vol. 59 , 22 December 1989 , CAMBRIDGE, NA US pages 1049 - 1059 LINTON, P-J. ET AL.; 'Primary antibody-forming cells and secondary B cells are generated from separate precursor cell populations' cited in the application * |
Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000020460A1 (en) * | 1998-10-05 | 2000-04-13 | Ludwig Institute For Cancer Research | Methods for producing human tumor antigen specific antibodies |
EP1392866A2 (en) * | 2001-05-14 | 2004-03-03 | Henry Hongjun Ji | Novel method for cloning variable domain sequences of immunological gene repertoire |
EP1392866A4 (en) * | 2001-05-14 | 2004-12-22 | Henry Hongjun Ji | Novel method for cloning variable domain sequences of immunological gene repertoire |
US10563254B2 (en) | 2007-01-23 | 2020-02-18 | Cambridge Enterprise Limited | Nucleic acid amplification and testing |
US11447821B2 (en) | 2007-01-23 | 2022-09-20 | Cambridge Enterprise Limited | Nucleic acid amplification and testing |
WO2012092376A3 (en) * | 2010-12-31 | 2012-12-06 | Short Jay M | Comprehensive monoclonal antibody generation |
CN103620405A (en) * | 2010-12-31 | 2014-03-05 | 生物蛋白有限公司 | Comprehensive monoclonal antibody generation |
CN103620405B (en) * | 2010-12-31 | 2016-05-25 | 生物蛋白有限公司 | Monoclonal antibody produces comprehensively |
US10670608B2 (en) | 2010-12-31 | 2020-06-02 | Bioatla, Llc | Comprehensive monoclonal antibody generation |
EP3961214A1 (en) * | 2010-12-31 | 2022-03-02 | BioAtla, Inc. | Comprehensive monoclonal antibody generation |
US11396717B2 (en) | 2010-12-31 | 2022-07-26 | Bioatla, Inc. | Comprehensive monoclonal antibody generation |
Also Published As
Publication number | Publication date |
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AU5673794A (en) | 1994-06-08 |
WO1994011507A3 (en) | 1994-07-07 |
EP0628076A1 (en) | 1994-12-14 |
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