Developing efficient hybridoma platform for therapeutic antibody discovery

Abstract

To provide development of an efficient hybridoma platform for therapeutic antibody discovery.SOLUTION: The present technology generally relates to improved methods for producing antibodies, antibody libraries, hybridomas, hybridoma libraries, and the like. For example, these methods increase the number of antigen-specific B cells produced, increase the number of hybridomas, and / or increase the number of monoclonal antibodies that can be produced in a given production cycle.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 894,660, filed August 30, 2019, the entire contents of which are incorporated herein by reference for all purposes. [Background technology]

[0002] background Monoclonal antibodies have been used for decades as important reagents in clinical diagnostics and are emerging as an important new class of therapeutic agents. Hybridoma technology is the most commonly used method for obtaining monoclonal antibodies. Monoclonal antibodies are secreted from hybridoma cells, which are generated by fusing normal antibody-producing splenic B cells with immortal myeloma cells or other immortal cells.

[0003] Hybridoma production has changed little since its inception several decades ago (Nature 256:495-497 (1975). A typical protocol for producing hybridomas involves: (i) immunizing an animal (e.g., a mouse, rat, or rabbit) with an antigen; (ii) harvesting antibody-producing B cells, typically from the spleen; (iii) fusing the B cells with a non-secreting myeloma cell line to form hybridomas; (iv) growing the hybridoma cells in a selective medium; (v) screening for cells that produce the desired antibody; and (vi) cloning the desired hybridoma(s) to obtain homogeneous cell lines that secrete antibodies. There is a need to generate increased numbers of antigen-specific antibodies and to improve the production of monoclonal antibodies and antibody libraries. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Nature 256:495-497(1975) Summary of the Invention [Means for solving the problem]

[0005] overview The present technology generally relates to improved methods for producing antibodies, antibody libraries, hybridomas, hybridoma libraries, etc., as well as compositions from various method steps. For example, these methods can increase the number of antigen-specific B cells produced, increase the number of hybridomas, and / or increase the number of monoclonal antibodies that can be made in a given production cycle. The methods described herein can be used to significantly increase the production of antibodies, antibody libraries, hybridomas, hybridoma libraries, etc., as well as compositions from various method steps, for use in treating various diseases. For example, the methods described herein can be applied to the generation of antibodies and / or hybridomas for treating cancer, immune disorders, inflammatory diseases, and any other disease treatable by antibody therapy. The methods described herein can be applied to the generation of antibodies and / or hybridomas against difficult targets. Difficult targets are antigens for which it is difficult to generate antibodies, for example, due to the small size of the targetable region, the importance of protein conformation, and / or the antigen being modified (e.g., by post-translational modifications such as glycosylation, phosphorylation, acetylation, and methylation). In some embodiments, the target antigen is a multi-pass transmembrane protein. In some embodiments, the multi-pass transmembrane protein is a G protein-coupled receptor (GPCR). In some embodiments, the multi-pass transmembrane protein is an ion pump, ion channel, or transporter.

[0006] In one aspect, a method for producing an antibody, an antibody library, a hybridoma, or a hybridoma library is provided. In embodiments, the method may include one or more of the following: (a) Injecting multiple animals with antigen; (b) harvesting B cells from each animal; (c) forming a hybridoma between the B cell and a fusion partner; and (d) screening the hybridomas for binding specificity to the antigen. In embodiments, at least one, preferably two or more of the following conditions may apply: (i) the animals are outbred; (ii) animals are injected at multiple sites; (iii) the animals are injected at least weekly, at least every 10 days, at least every 2 weeks, or at some other frequency with a longer period between injections; (iv) animals are injected for 6 to 15 weeks; (v) using multiple adjuvants such that each animal is injected with a single adjuvant and at least some animals are injected with different adjuvants; (vi) enriching B cells prior to step (c); and / or (vii) Using a fusion partner engineered to express both surface and secreted IgG.

[0007] In one aspect, a method for producing an antibody, an antibody library, a hybridoma, or a hybridoma library is provided. In embodiments, the method may include one or more of the following: (a) injecting one or more animals with an antigen; (b) harvesting B cells from each animal; (c) forming a hybridoma between the B cell and a fusion partner; and (d) screening the hybridomas for binding specificity to the antigen. In embodiments, at least one, preferably two or more of the following conditions may apply: (i) the animals are outbred; (ii) animals are injected at multiple sites; (iii) the animals are injected at least weekly, at least every 10 days, at least every 2 weeks, or at some other frequency with a longer period between injections; (iv) animals are injected for 6 to 15 weeks; (v) using multiple adjuvants such that each animal is injected with a single adjuvant and different animals are injected with different adjuvants; (vi) enriching B cells prior to step (c); and / or (vii) Using a fusion partner engineered to express both surface and secreted IgG.

[0008] In embodiments, at least one, preferably two or more of the following conditions may apply: (i) the animals are outbred; (ii) animals are injected at multiple sites; (iii) the animals are injected at least weekly, at least every 10 days, at least every 2 weeks, or at some other frequency with a longer period between injections; (iv) animals are injected for 6 to 15 weeks; (v) using multiple adjuvants such that each animal is injected with a single adjuvant and different animals are injected with different adjuvants; and / or (vi) B cells are enriched prior to step (c).

[0009] In embodiments, B cells may be harvested from draining lymph nodes.

[0010] In embodiments, the method may include injecting two or more animals with the antigen. In embodiments, the method may include injecting three or more animals with the antigen. In embodiments, the method may include injecting four or more animals with the antigen. In embodiments, the method may include injecting five or more animals with the antigen.

[0011] In multiple embodiments, two of conditions (i) to (vii) may apply. In multiple embodiments, three of conditions (i) to (vii) may apply. In multiple embodiments, four of conditions (i) to (vii) may apply. In multiple embodiments, five of conditions (i) to (vii) may apply. In multiple embodiments, six of conditions (i) to (vii) may apply. In multiple embodiments, seven of conditions (i) to (vii) may apply. In multiple embodiments, two of conditions (i) to (vi) may apply. In multiple embodiments, three of conditions (i) to (vi) may apply. In multiple embodiments, four of conditions (i) to (vi) may apply. In multiple embodiments, five of conditions (i) to (vi) may apply. In multiple embodiments, six of conditions (i) to (vi) may apply.

[0012] In embodiments, the method may include: (a) Injecting multiple outbred rats with antigen at multiple sites in each rat; (b) repeat injections every 2 weeks for at least 6 weeks; (c) harvesting immune cells from one or more draining lymph nodes of each rat; (d) depleting non-B cells from the immune cells by negative selection to form an enriched B cell sample; (e) contacting the enriched B cell sample with a plurality of fusion partners to form hybridomas between the B cells and the fusion partners; and (f) screening hybridomas for specificity for the antigen;

[0013] In embodiments, the method may include: (a) injecting one or more outbred rats with antigen at multiple sites in each rat; (b) repeat injections every 2 weeks for at least 6 weeks; (c) harvesting immune cells from one or more draining lymph nodes of each rat; (d) depleting non-B cells from the immune cells by negative selection to form an enriched B cell sample; (e) contacting the enriched B cell sample with a plurality of fusion partners to form hybridomas between the B cells and the fusion partners; and (f) screening hybridomas for specificity for the antigen;

[0014] In embodiments, the animal may be a rat. In embodiments, the animal may be an outbred rat. Examples of outbred rats include, but are not limited to, Sprague Dawley, Long-Evans, Sentinel, Wistar, Wistar Han, and Holtzmann rats. In embodiments, the animal may be a transgenic animal. In embodiments, the animal may be a transgenic rat.

[0015] In some embodiments, an animal can be injected at multiple sites. In some embodiments, the multiple sites can be near draining lymph nodes. In some embodiments, the multiple sites can include one or more of the back, shoulder, intraperitoneal cavity, base of tail, and ankle.

[0016] In some embodiments, the amount of antigen injected at each site may be between 0.1 μg and 300 μg, and in some embodiments, the amount of antigen injected at each site may be between 0.5 μg and 200 μg.

[0017] In embodiments, the method may include injecting two or more outbred rats with the antigen. In embodiments, the method may include injecting three or more outbred rats with the antigen. In embodiments, the method may include injecting four or more outbred rats with the antigen. In embodiments, the method may include injecting five or more outbred rats with the antigen.

[0018] In embodiments, animals may be injected every two weeks. In embodiments, animals may be injected no more than once a week. In embodiments, animals may be injected no more than once every two weeks. In embodiments, animals may be injected every 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 20, 21, 22, 23, 24, 25, 26, 27, 28 days or more for 6 to 19 weeks. In embodiments, animals may be injected for 6 to 15 weeks. The times of injection include all values and subranges encompassed by the recited ranges, including the endpoints.

[0019] In some embodiments, multiple adjuvants can be used, and each animal (or subset of animals) can be injected with a different adjuvant. For example, animals can be divided into groups, with each member of a group receiving a single adjuvant or combination of adjuvants, each adjuvant or combination different from that received by at least some of the other groups. In some embodiments, the multiple adjuvants can include, but are not limited to, complete Freund's adjuvant (CFR), Ribi, and / or a TLR agonist cocktail. In some embodiments, the CFR can be mixed mechanically.

[0020] In embodiments, B cells can be enriched before hybridoma formation. In embodiments, enrichment can include contacting cells harvested from the draining lymph node with a binder. In embodiments, B cells can be enriched by negative selection. In embodiments, the binder can be specific for a molecule associated with a cell that is not a B cell. In embodiments, the binder can be specific for a molecule expressed on the surface of a cell that is not a B cell. In embodiments, the binder can be specific for a molecule associated with a B cell. In embodiments, the binder can be an antibody. In embodiments, magnetic separation can be used.

[0021] In some embodiments, the fusion partner may be a cell engineered to express both surface and secreted IgG. In some embodiments, the fusion partner may be an Sp2ab fusion partner. In some embodiments, screening the hybridomas may include identifying hybridomas that express antibodies specific to the antigen. In some embodiments, screening the hybridomas may include FACS sorting for expression of IgG antibodies specific to the antigen. In an embodiment of the present invention, for example, the following items are provided: (Item 1) 1. A method for producing an antibody library, comprising: (a) injecting one or more animals with an antigen; (b) harvesting B cell-containing draining lymph nodes from each animal; (c) forming a hybridoma between each B cell and a fusion partner; and (d) screening the hybridomas for binding specificity to the antigen. Including, The following conditions: (i) the animal is an outbred animal; (ii) the animal is injected at multiple sites; (iii) the animals are injected every two weeks; (iv) the animals are injected for 6 to 15 weeks; (v) using multiple adjuvants such that different animals are injected with different adjuvants; (vi) enriching B cells prior to step (c); and / or (vii) Use a fusion partner engineered to express both surface and secreted IgG. At least two of the following methods are applied: (Item 2) Item 1. The method according to item 1, wherein two of conditions (i) to (vii) apply. (Item 3) Item 1. The method according to item 1, wherein three of the conditions (i) to (vii) apply. (Item 4) Item 1. The method according to item 1, wherein four of the conditions (i) to (vii) apply. (Item 5) Item 1. The method according to item 1, wherein five of the conditions (i) to (vii) apply. (Item 6) Item 1. The method according to item 1, wherein six of the conditions (i) to (vii) apply. (Item 7) Item 1. The method according to item 1, wherein seven of conditions (i) to (vii) apply. (Item 8) Item 1. The method according to item 1, wherein at least two of conditions (i) to (vi) apply. (Item 9) 2. The method of claim 1, wherein step (a) comprises injecting two or more animals with the antigen. (Item 10) 2. The method of claim 1, wherein step (a) comprises injecting three or more animals with the antigen. (Item 11) 2. The method of claim 1, wherein step (a) comprises injecting four or more animals with the antigen. (Item 12) 2. The method of claim 1, wherein step (a) comprises injecting five or more animals with the antigen. (Item 13) 13. The method of any one of items 1 to 12, wherein the animal is a rat. (Item 14) 14. The method of any one of items 1 to 13, wherein the multiple sites are sites near draining lymph nodes. (Item 15) 15. The method of any one of items 1 to 14, wherein the multiple sites include one or more of the back, shoulder, abdominal cavity, base of the tail, and ankle. (Item 16) 16. The method of any one of items 1 to 15, wherein the animal is injected at multiple sites and the amount of antigen injected at each site is between 0.1 μg and 300 μg. (Item 17) 17. The method of any one of items 1 to 16, wherein the animal is injected at multiple sites and the amount of antigen injected at each site is between 0.5 μg and 200 μg. (Item 18) 18. The method of any one of items 1 to 17, wherein the animal is injected no more than once a week. (Item 19) 19. The method of any one of items 1 to 18, wherein the animal is injected no more than once every two weeks. (Item 20) 20. The method of any one of items 1 to 19, wherein the animal is injected every 6 to 15 weeks. (Item 21) 21. The method of any one of items 1 to 20, wherein the multiple adjuvants comprise complete Freund's adjuvant, Ribi, and / or a TLR agonist cocktail. (Item 22) 22. The method of any one of items 1 to 21, wherein the B cells are enriched by negative selection. (Item 23) 23. The method of any one of items 1 to 22, wherein the fusion partner is an Sp2ab fusion partner. (Item 24) 24. The method of any one of items 1 to 23, wherein screening the hybridomas comprises FACS sorting for expression of IgG antibodies specific for the antigen. (Item 25) 1. A method for producing an antibody library, comprising: (a) injecting one or more outbred rats with antigen at multiple sites in each rat; (b) repeating said injection every two weeks for at least six weeks; (c) harvesting immune cells from one or more draining lymph nodes of each rat; (d) depleting non-B cells from said immune cells by negative selection to form an enriched B cell sample; (e) contacting the enriched B cell sample with a plurality of fusion partners to form hybridomas between each B cell and the fusion partner; and (f) screening said hybridomas for specificity for said antigen. (Item 26) 26. The method of claim 25, wherein the multiple sites are sites near draining lymph nodes. (Item 27) 27. The method of claim 25 or 26, wherein the multiple sites include one or more of the back, shoulder, abdominal cavity, base of the tail, and ankle. (Item 28) 28. The method of any one of items 25 to 27, wherein the amount of antigen injected at each site is between 0.1 μg and 300 μg. (Item 29) 29. The method according to item 28, wherein the amount of antigen injected at each site is between 0.5 μg and 200 μg. (Item 30) 30. The method of any one of items 25 to 29, wherein the animal is injected no more than once every two weeks. (Item 31) 31. The method of any one of items 25 to 30, wherein the animal is injected every 6 to 10 weeks. (Item 32) 32. The method of any one of items 25 to 31, wherein the fusion partner is an Sp2ab fusion partner. (Item 33) 33. The method of any one of items 25 to 32, wherein screening the hybridomas comprises FACS sorting for expression of IgG antibodies specific for the antigen. (Item 34) 34. The method of any one of items 21 to 33, wherein step (a) comprises injecting two or more animals with the antigen. (Item 35) 34. The method of any one of items 21 to 33, wherein step (a) comprises injecting three or more animals with the antigen. (Item 36) 34. The method of any one of items 21 to 33, wherein step (a) comprises injecting four or more animals with the antigen. (Item 37) 34. The method of any one of items 21 to 33, wherein step (a) comprises injecting five or more animals with the antigen. (Item 38) 38. An antibody library prepared using the method of any one of items 1 to 37. (Item 39) 38. A hybridoma library prepared using the method of any one of items 1 to 37. [Brief explanation of the drawings]

[0022] [Figure 1A] Shown is the difference in IgG serum titers when Sprague Dawley rats (SD rats) versus Balb / c mice are used for antibody production against antigen A (target A). The homology of target A in mice versus rats is 98%. *p<0.05 vs. mouse.

[0023] [Figure 1B] The difference in IgG serum titers is shown when Sprague Dawley rats (SD rats) versus C57 Black 6 (C57 / BL6) mice are used for antibody production against antigen B (target B). *p<0.05 vs. mice.

[0024] [Figure 2A] FIG. 1 is a diagram of a rat showing an example of an antigen injection site for immunization.

[0025] [Figure 2B] Antigen-specific antibody titers against antigens C (target C) and D (target D) using single-site injection or multiple-site injection for each animal are shown. *p<0.05 vs. single injection.

[0026] [Figure 3A] Figure 3A shows an exemplary protocol for injecting animals with antigen and the expected antigen-specific Ig (IgM and IgG) responses based on that protocol. Background serum titers are performed before the first injection ("titer check bleed"). Animals are injected with antigen in adjuvant at week 0 ("prime"), then injected with antigen in PBS once every two weeks until week 6 ("boost"). Serum titers are measured again after the final injection.

[0027] [Figure 3B] Figures 3B and 3C compare the standard antigen injection protocol (two injections per week for 9 weeks) with the improved protocol shown in Figure 3A. The improved protocol increases antibody titers against antigen E (target E) when administered in Ribi adjuvant (Figure 3B) or TLR adjuvant (Figure 3C). [Figure 3C] Figures 3B and 3C compare the standard antigen injection protocol (two injections per week for 9 weeks) with the improved protocol shown in Figure 3A. The improved protocol increases antibody titers against antigen E (target E) when administered in Ribi adjuvant (Figure 3B) or TLR adjuvant (Figure 3C).

[0028] [Figure 4A] FIG. 10 is a diagram of a rat showing an example of a draining lymph node near the injection site.

[0029] [Figure 4B] Figure 1 shows the difference in the number of antigen (target D)-specific hybridoma clones when cells are harvested from lymph nodes compared to spleens. The increase in IgG+ hybridomas from LNs applies to rats (as opposed to mice).

[0030] [Figure 5A] Antigen-specific IgG serum titers against antigen J are shown. CFA adjuvant was used and mixed either mechanically or by hand ("syringe mixed"). *p<0.01 vs. syringe mixed.

[0031] [Figure 5B] Figures 5B and 5C show antigen-specific IgG serum titers against antigen F ("Target F", Figure 5B) or antigen G ("Target G", Figure 5C) when CFA, Ribi, or TLR was used as an adjuvant. *p<0.05 vs. other conditions. [Figure 5C] Figures 5B and 5C show antigen-specific IgG serum titers against antigen F ("Target F", Figure 5B) or antigen G ("Target G", Figure 5C) when CFA, Ribi, or TLR was used as an adjuvant. *p<0.05 vs. other conditions.

[0032] [Figure 6] The numbers of IgG-positive hybridoma clones derived from whole lymph nodes ("unseparated"), cells remaining after depletion of non-B cells from lymphocyte harvested cells ("B cells"), or cells remaining after depletion of IgM-positive B cells from enriched B cells are shown.

[0033] [Figure 7A] FIG. 7A shows the number of antigen H ("target H")-specific hybridomas generated using either P3X63AgU.1 ("PU.1"; ATCC) or Sp2ab (Enzo Abeome DiSH) as the fusion partner.

[0034] [Figure 7B] FIG. 7B shows the FACS sorting profile of the SP2ab hybridoma.

[0035] [Figure 7C] Figures 7C and 7D show the percentage of antigen-specific hybridomas generated using either the PU.1 or Sp2ab fusion partners. [Figure 7D] Figures 7C and 7D show the percentage of antigen-specific hybridomas generated using either the PU.1 or Sp2ab fusion partners. DETAILED DESCRIPTION OF THE INVENTION

[0036] Detailed Description After reading this description, it will be apparent to one skilled in the art how to implement the present disclosure in various alternative embodiments and alternative applications. However, not all of the various embodiments of the present technology are described herein. It will be understood that the embodiments presented herein are presented by way of example only and not limitation. Therefore, this detailed description of various alternative embodiments should not be construed as limiting the scope or breadth of the present disclosure described herein.

[0037] Before the present technology is disclosed and described, it is to be understood that the embodiments described below are not limited to particular compositions, methods of preparing such compositions, or uses thereof, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0038] The detailed description is divided into various sections solely for the convenience of the reader, and disclosure found in any section may be combined with that of another section. Titles or subtitles may be used herein for the convenience of the reader and are not intended to affect the scope of the disclosure.

[0039] definition Unless otherwise defined, 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 disclosure belongs. In this specification and the claims that follow, reference will be made to several terms that shall be defined to have the following meanings.

[0040] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0041] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not occur.

[0042] The term "about," when used before a numerical designation including a range, e.g., temperature, time, amount, concentration, etc., indicates an approximation that may vary by (+) or (-) 10%, 5%, 1%, or any subrange or subvalue therebetween. Preferably, when used in reference to a dose, the term "about" means that the dose may vary by + / - 10%.

[0043] As used herein, the term "near" is intended to mean a location within a short physical distance of an object or point in space, or a location within a short distance. In some embodiments, "near" may be between about 0 mm and about 50 mm. In some embodiments, "near" may be between about 0 mm and about 40 mm. In some embodiments, "near" may be between about 0 mm and about 30 mm. In some embodiments, "near" may be between about 0 mm and about 20 mm. In some embodiments, "near" may be between about 0 mm and about 10 mm. In some embodiments, "near" may be less than about 1 mm. In some embodiments, "near" may be less than about 5 mm. In some embodiments, "near" may be less than about 1 cm. In some embodiments, "near" may be less than about 2 cm. In some embodiments, "near" may be less than about 5 cm. In some embodiments, "near" may be about 1 mm. In some embodiments, "near" may be about 2 mm. In some embodiments, "near" may be about 3 mm. In some embodiments, "near" may be about 4 mm. In some embodiments, "near" may be about 5 mm. In some embodiments, "near" may be about 1 cm. In some embodiments, "near" may be about 2 cm. In some embodiments, "near" may be about 3 cm. In some embodiments, "near" may be about 4 cm. The distance may be any value or subrange within the recited range, including the endpoints.

[0044] The terms "comprising" or "comprises" are intended to mean that compositions and methods include the recited elements, but do not exclude others. "Consisting essentially of," when used to define compositions and methods, is intended to mean excluding other elements that are essential to the combination for the purpose described. Thus, a composition consisting essentially of the elements defined herein does not exclude other materials or steps that do not materially affect the basic and novel characteristic(s) of the claimed technology. "Consisting of" is intended to mean excluding more than trace amounts of other components and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0045] The term "antibody" is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0046] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., each individual antibody within the population is identical and / or binds to the same epitope, except for possible variant antibodies, including, for example, naturally occurring mutations or mutations that arise during production of a monoclonal antibody preparation, of which such variants will generally be present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring production of the antibody by any particular method.

[0047] As used herein, the term "fusion partner" refers to a cell that can combine with (fuse with) a B cell to form a hybridoma. Generally, the fusion partner is a myeloma cell.

[0048] As used herein, the term "outbred" refers to an animal that is genetically distinct from other animals of the same species. In contrast, "inbred" refers to an animal that is genetically identical (or nearly identical) to others in its lineage due to inbreeding.

[0049] The term "fusion partner" as used herein refers to a fusion partner for producing a hybridoma. Methods and cells for producing hybridomas from various species are well known in the art. Generally, the fusion partner is a plurality of myeloma cells. The fusion partner may be any suitable cell or cell line for producing a hybridoma, such as a myeloma. The fusion partner may be of mammalian origin. The mammalian origin may be a primate, human, rat, mouse, rodent, or any other species.

[0050] method In one aspect, a method for producing an antibody is provided. In one aspect, a method for producing an antibody library is provided. In one aspect, a method for producing a hybridoma is provided. In one aspect, a method for producing a hybridoma library is provided.

[0051] In embodiments, the method may include one or more of the following: (a) injecting one or more animals with an antigen; (b) harvesting B cells from each animal, e.g., from B cell-containing draining lymph nodes; (c) forming a hybridoma between one or more, or each, B cell(s) and a fusion partner(s); and (d) screening one or more hybridomas for binding specificity to the antigen.

[0052] In embodiments, at least one, or preferably at least two of the following conditions may apply: (i) the animals are outbred; (ii) animals are injected at multiple sites; (iii) the animals are injected at a frequency described herein no more than once, e.g., more than once every two weeks; (iv) the animals are injected at a frequency described herein for a period of about 6 weeks to about 15 weeks; (v) multiple adjuvants are used such that different animals or groups of animals are injected with different adjuvants compared to other animals or groups of animals; (vi) enriching B cells prior to step (c); and / or (vii) Using a fusion partner engineered to express both surface and secreted IgG.

[0053] In some embodiments, at least two of conditions (a)-(d) may apply. In some embodiments, at least three of conditions (a)-(d) may apply. In some embodiments, four of conditions (a)-(d) may apply. In some embodiments, one or more of conditions (a)-(d) may be explicitly excluded.

[0054] In embodiments, at least three of conditions (i) through (vii) may apply. In embodiments, at least four of conditions (i) through (vii) may apply. In embodiments, at least five of conditions (i) through (vii) may apply. In embodiments, at least six of conditions (i) through (vii) may apply. In embodiments, two of conditions (i) through (vii) may apply. In embodiments, three of conditions (i) through (vii) may apply. In embodiments, four of conditions (i) through (vii) may apply. In embodiments, five of conditions (i) through (vii) may apply. In embodiments, six of conditions (i) through (vii) may apply. In embodiments, seven of conditions (i) through (vii) may apply. In embodiments, one or more of conditions (i) through (vii) may be explicitly excluded. In embodiments, condition (i) is explicitly excluded. In embodiments, condition (ii) is explicitly excluded. In embodiments, condition (iii) is expressly excluded. In embodiments, condition (iv) is expressly excluded. In embodiments, condition (v) is expressly excluded. In embodiments, condition (vi) is expressly excluded. In embodiments, condition (vii) is expressly excluded.

[0055] In embodiments, the method can include: (a) injecting one or more outbred rats with antigen at multiple sites in each rat; (b) repeating said injection every two weeks for at least six weeks; (c) harvesting immune cells from one or more draining lymph nodes of each rat; (d) depleting non-B cells from the immune cells by negative selection to form an enriched B cell sample; (e) contacting the enriched B cell sample with a plurality of fusion partners to form hybridomas between each B cell and the fusion partner; and (f) screening said hybridomas for specificity for said antigen.

[0056] In some embodiments, the animal may be an outbred animal. In some embodiments, the animal may be a mammal. In some embodiments, the animal may be a rodent. In some embodiments, the rodent may be a rabbit, guinea pig, rat, hamster, mouse, etc. In some embodiments, the animal may be a rat. In some embodiments, the animal may be an outbred rat. Examples of outbred rats include, but are not limited to, Sprague Dawley, Long-Evans, Sentinel, CD® IGS (Charles River), CD® Hairless, Wistar, Wistar Han, and Holtzman rats.

[0057] In some embodiments, the animal may be a mouse. In some embodiments, the animal may be an outbred mouse. Examples of outbred mouse strains include, but are not limited to, Black Swiss, CD-1® IGS (e.g., from Charles River), CF-1, CFW, ORL Sencar, SKH1-Elite, Sentinel, and Diversity Outbred (Jackson Laboratory).

[0058] In embodiments, the method may include injecting two or more outbred rats with the antigen at multiple sites within each rat. In embodiments, the method may include injecting three or more outbred rats with the antigen at multiple sites within each rat. In embodiments, the method may include injecting four or more outbred rats with the antigen at multiple sites within each rat. In embodiments, the method may include injecting five or more outbred rats with the antigen at multiple sites within each rat.

[0059] In some embodiments, the animal may be injected at one or more sites. In some embodiments, the animal is injected at multiple sites. In some embodiments, the one or more sites may be near a draining lymph node. In some embodiments, the one or more sites may include one or more of the back, shoulder, intraperitoneal, base of the tail, ankle, and intravenous.

[0060] In some embodiments, the amount of antigen injected at each site may be about 0.1 μg to about 300 μg. In some embodiments, the amount of antigen injected at each site may be 0.1 μg to 200 μg. In some embodiments, the amount of antigen injected at each site may be 0.1 μg to 100 μg. In some embodiments, the amount of antigen injected at each site may be 0.1 μg to 50 μg. In some embodiments, the amount of antigen injected at each site may be 0.1 μg to 25 μg. In some embodiments, the amount of antigen injected at each site may be 0.1 μg to 10 μg. In some embodiments, the amount of antigen injected at each site may be 0.5 μg to 200 μg. In some embodiments, the amount of antigen injected at each site may be 0.5 μg to 100 μg. In some embodiments, the amount of antigen injected at each site may be 0.5 μg to 50 μg. In several embodiments, the amount of antigen injected at each site may be 0.5 μg to 25 μg. In several embodiments, the amount of antigen injected at each site may be 0.5 μg to 10 μg. In several embodiments, the amount of antigen injected at each site may be 1 μg to 300 μg. In several embodiments, the amount of antigen injected at each site may be 1 μg to 200 μg. In several embodiments, the amount of antigen injected at each site may be 1 μg to 100 μg. In several embodiments, the amount of antigen injected at each site may be 1 μg to 50 μg. In several embodiments, the amount of antigen injected at each site may be 1 μg to 25 μg. In several embodiments, the amount of antigen injected at each site may be 1 μg to 10 μg. In several embodiments, the amount of antigen injected at each site may be 5 μg to 300 μg. In some embodiments, the amount of antigen injected at each site may be 5 μg to 200 μg. In some embodiments, the amount of antigen injected at each site may be 5 μg to 100 μg. In some embodiments, the amount of antigen injected at each site may be 5 μg to 50 μg. In some embodiments, the amount of antigen injected at each site may be 5 μg to 25 μg. In some embodiments, the amount of antigen injected at each site may be 5 μg to 10 μg.The amount can be any value or subrange within the recited range, including the endpoints.

[0061] In embodiments, animals may be injected at one site or multiple sites on a single day or period occurring every one to four weeks or more, preferably every week or more, every ten days or more, every two weeks or more, every three weeks or more, every four weeks or more, etc. In embodiments, one or more sites may be injected at different times, but each site is injected no more frequently than every one to four weeks, preferably no more frequently than every two weeks, etc. In some embodiments, animals are injected no more than once a week. In some embodiments, animals are injected no more than once every two weeks.

[0062] In embodiments, animals may receive injections at the frequency described herein for 6 to 15 weeks. In embodiments, animals may be injected between 7 and 15 weeks. In embodiments, animals may be injected between 8 and 15 weeks. In embodiments, animals may be injected between 9 and 15 weeks. In embodiments, animals may be injected between 10 and 15 weeks. In embodiments, animals may be injected between 11 and 15 weeks. In embodiments, animals may be injected between 12 and 15 weeks. In embodiments, animals may be injected between 13 and 15 weeks. In embodiments, animals may be injected between 14 and 15 weeks.

[0063] In embodiments, an animal may be initially injected at least one site with a first composition comprising an antigen and an adjuvant, and then every two weeks with a second composition comprising an antigen at the same or a different site. In embodiments, the animal may be injected no more than once every two weeks. In embodiments, the animal may be injected at the same site no more than once every two weeks. In some embodiments, the second composition does not include an adjuvant. In some embodiments, the second composition includes an adjuvant. In some embodiments, lymph nodes may be harvested from the animal after the second, third, fourth, fifth, or sixth injection. A second, third, fourth, fifth, or sixth injection can refer to a second, third, fourth, fifth, or sixth injection of the animal at the same site(s) or a second, third, fourth, fifth, or sixth injection at a different site.

[0064] In embodiments, the lymph nodes may be harvested from the animal, for example, between 6 and 10 weeks after the initial injection. In embodiments, the lymph nodes may be harvested from the animal between 6 and 8 weeks after the initial injection. In embodiments, the lymph nodes may be harvested from the animal about 6 weeks after the initial injection. In embodiments, the lymph nodes may be harvested from the animal about 7 weeks after the initial injection. In embodiments, the lymph nodes may be harvested from the animal about 8 weeks after the initial injection. In embodiments, the lymph nodes may be harvested from the animal about 9 weeks after the initial injection. In embodiments, the lymph nodes may be harvested from the animal about 10 weeks after the initial injection.

[0065] In some embodiments, multiple adjuvants may be used, and each animal (or subset of animals) may be injected with a different adjuvant. In some embodiments, the multiple adjuvants may include, for example, complete Freund's adjuvant (CFR), Ribi, and / or a TLR (Toll-like receptor) agonist cocktail. In some embodiments, the adjuvant may be incomplete Freund's adjuvant. In some embodiments, the adjuvant may be TiterMax® (a water-in-oil emulsion containing block copolymer CRL-8941, squalene, and a particulate stabilizer).

[0066] Some adjuvants are mixed to form an emulsion. Without being bound by theory, it is believed that mechanical mixing of the adjuvants results in more consistent emulsification and improved results compared to manual mixing (e.g., using a syringe). In some embodiments, the adjuvants may be mechanically mixed. In some embodiments, the mechanically mixed adjuvant may be CFR.

[0067] In some embodiments, B cells may be enriched prior to hybridoma formation. In some embodiments, enrichment may include contacting cells harvested from the draining lymph node with a binder. In some embodiments, B cells may be enriched by negative selection. In some embodiments, the binder may be specific for a molecule associated with a cell that is not a B cell. In some embodiments, the binder may be specific for a molecule expressed on the surface of a cell that is not a B cell. In some embodiments, B cells may be enriched by positive selection. In some embodiments, the binder may be specific for a molecule associated with a B cell. In some embodiments, the binder may be specific for a molecule expressed on the surface of a B cell (e.g., a B cell-specific cell surface receptor).

[0068] A binding agent can be any agent that binds to a molecule of interest. In some embodiments, the binding agent can be an antibody or a portion thereof. In some embodiments, the binding agent can be a fusion protein, an aptamer, a ligand, or a receptor.

[0069] In some embodiments, magnetic separation may be used. In some embodiments, magnetic beads may be used. The magnetic beads may be bound to or otherwise associated with a binding agent.

[0070] In embodiments, the fusion partner is engineered to express both surface and secreted IgG. See, e.g., U.S. Patent No. 7,148,040, which is incorporated herein by reference in its entirety for all of its materials, methods, and teachings. In embodiments, the fusion partner is an Sp2ab fusion partner. Sp2ab myeloma fusion partners are available from Abeome Corporation or Enzo Life. Available from Sciences.

[0071] In some embodiments, screening the hybridomas may include identifying hybridomas that express antibodies specific to the antigen, hi some embodiments, screening the hybridomas may include FACS sorting for expression of IgG antibodies specific to the antigen.

[0072] In embodiments, the method may increase antibody and / or hybridoma production by at least 50% compared to production without at least two conditions selected from (i)-(vii). In embodiments, the method may increase antibody and / or hybridoma production by at least two-fold compared to production without at least two conditions selected from (i)-(vii). In embodiments, the method may increase antibody and / or hybridoma production by at least three-fold compared to production without at least two conditions selected from (i)-(vii). In embodiments, the method may increase antibody and / or hybridoma production by at least four-fold compared to production without at least two conditions selected from (i)-(vii). In embodiments, the method may increase antibody and / or hybridoma production by at least five-fold compared to production without at least two conditions selected from (i)-(vii). In embodiments, the method may increase antibody and / or hybridoma production by at least 6-fold compared to production without at least two conditions selected from (i)-(vii). In embodiments, the method may increase antibody and / or hybridoma production by at least 7-fold compared to production without at least two conditions selected from (i)-(vii). In embodiments, the method may increase antibody and / or hybridoma production by at least 8-fold compared to production without at least two conditions selected from (i)-(vii). In embodiments, the method may increase antibody and / or hybridoma production by at least 9-fold compared to production without at least two conditions selected from (i)-(vii). In embodiments, the method may increase antibody and / or hybridoma production by at least 10-fold compared to production without at least two conditions selected from (i)-(vii). In several embodiments, the method may increase antibody and / or hybridoma production by at least 20-fold compared to production without at least two conditions selected from (i) to (vii).In embodiments, the method may increase antibody and / or hybridoma production by at least 30-fold compared to production without at least two conditions selected from (i)-(vii). In embodiments, the method may increase antibody and / or hybridoma production by at least 40-fold compared to production without at least two conditions selected from (i)-(vii). In embodiments, the method may increase antibody and / or hybridoma production by at least 50-fold compared to production without at least two conditions selected from (i)-(vii). In embodiments, the method may increase antibody and / or hybridoma production by at least 60-fold compared to production without at least two conditions selected from (i)-(vii). In embodiments, the method may increase antibody and / or hybridoma production by at least 70-fold compared to production without at least two conditions selected from (i)-(vii). In embodiments, the method may increase antibody and / or hybridoma production by at least 80-fold compared to production without at least two conditions selected from (i)-(vii). In embodiments, the method may increase antibody and / or hybridoma production by at least 90-fold compared to production without at least two conditions selected from (i)-(vii). In embodiments, the method may increase antibody and / or hybridoma production by more than 100-fold compared to production without at least two conditions selected from (i)-(vii).

[0073] In embodiments, obtaining hybridoma clones from, e.g., lymph nodes according to the methods described herein produces up to about 15-fold more hybridoma clones than obtaining hybridoma clones from spleen tissue, e.g., by an otherwise similar or identical method. See, e.g., the methods compared in Example 4. In embodiments, obtaining hybridoma clones from, e.g., lymph nodes according to the methods described herein produces about 2-fold to about 15-fold more hybridoma clones than obtaining hybridoma clones from spleen tissue, e.g., by an otherwise similar or identical method. In embodiments, obtaining hybridoma clones from, e.g., lymph nodes according to the methods described herein produces about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14-fold, or about 15-fold more hybridoma clones than obtaining hybridoma clones from spleen tissue, e.g., by an otherwise similar or identical method. The amount can be any value or subrange within the recited range, including the endpoints. In some embodiments, the lymph node is from a rat, eg, a wild-type rat or a transgenic rat.

[0074] In embodiments, mechanical mixing of the adjuvant can result in an antigen-specific serum IgG titer that is two to four times higher than that obtained by syringe mixing of the adjuvant. In embodiments, mechanical mixing of the adjuvant can result in an antigen-specific serum IgG titer that is two times higher than that obtained by syringe mixing of the adjuvant. In embodiments, mechanical mixing of the adjuvant can result in an antigen-specific serum IgG titer that is three times higher than that obtained by syringe mixing of the adjuvant. In embodiments, mechanical mixing of the adjuvant can result in an antigen-specific serum IgG titer that is four times higher than that obtained by syringe mixing of the adjuvant.

[0075] In embodiments, the antigen-specific IgG titer when using complete Freund's adjuvant (CFA) may be 10-fold or more higher than when using a different adjuvant, such as Ribi adjuvant or a TLR agonist cocktail adjuvant. In embodiments, the antigen-specific IgG titer when using CFA may be 2-fold to 50-fold higher. In embodiments, the antigen-specific IgG titer when using CFA may be 2-fold to 25-fold higher. In embodiments, the antigen-specific IgG titer when using CFA may be 2-fold to 20-fold higher. In embodiments, the antigen-specific IgG titer when using CFA may be 2-fold to 15-fold higher. In embodiments, the antigen-specific IgG titer when using CFA may be 2-fold to 10-fold higher. In embodiments, the antigen-specific IgG titer when using CFA may be 10-fold to 50-fold higher. In embodiments, the antigen-specific IgG titer when using CFA may be 10-fold to 25-fold higher. In embodiments, antigen-specific IgG titers can be 10- to 20-fold higher using CFA. The amount can be any value or subrange within the recited range, including the endpoints.

[0076] In embodiments, antigen-specific IgG titers using Ribi adjuvants may be 10-fold or more higher than when using a different adjuvant, such as a TLR agonist cocktail adjuvant. In embodiments, antigen-specific IgG titers using Ribi adjuvants may be 2-fold to 50-fold higher. In embodiments, antigen-specific IgG titers using Ribi adjuvants may be 2-fold to 25-fold higher. In embodiments, antigen-specific IgG titers using Ribi adjuvants may be 2-fold to 20-fold higher. In embodiments, antigen-specific IgG titers using Ribi adjuvants may be 2-fold to 15-fold higher. In embodiments, antigen-specific IgG titers using Ribi adjuvants may be 2-fold to 10-fold higher. In embodiments, antigen-specific IgG titers using Ribi adjuvants may be 10-fold to 50-fold higher. In embodiments, antigen-specific IgG titers can be 10- to 25-fold higher with Ribi adjuvant. In embodiments, antigen-specific IgG titers can be 10- to 20-fold higher with Ribi adjuvant. The amount can be any value or subrange within the recited range, including the endpoints.

[0077] In some embodiments, the antigen-specific IgG titer may be 10-fold or more higher when a TLR agonist cocktail adjuvant is used compared to when a different adjuvant, such as Ribi adjuvant or CFA, is used. In some embodiments, the antigen-specific IgG titer may be 2-fold to 50-fold higher when a TLR agonist cocktail adjuvant is used. In some embodiments, the antigen-specific IgG titer may be 2-fold to 25-fold higher when a TLR agonist cocktail adjuvant is used. In some embodiments, the antigen-specific IgG titer may be 2-fold to 20-fold higher when a TLR agonist cocktail adjuvant is used. In some embodiments, the antigen-specific IgG titer may be 2-fold to 15-fold higher when a TLR agonist cocktail adjuvant is used. In some embodiments, the antigen-specific IgG titer may be 2-fold to 10-fold higher when a TLR agonist cocktail adjuvant is used. In embodiments, antigen-specific IgG titers may be 10- to 50-fold higher when using a TLR agonist cocktail adjuvant. In embodiments, antigen-specific IgG titers may be 10- to 25-fold higher when using a TLR agonist cocktail adjuvant. In embodiments, antigen-specific IgG titers may be 10- to 20-fold higher when using a TLR agonist cocktail adjuvant. The amount may be any value or subrange within the recited range, including the endpoints.

[0078] In some embodiments, the use of IgM-depleted enriched LN B cells may produce about 10-fold to about 100-fold more IgG-expressing hybridoma clones than when enriched LN B cells are used. In some embodiments, the use of IgM-depleted enriched LN B cells produces about 10-fold to about 75-fold more IgG-expressing hybridoma clones. The amounts may be any value or subrange within the recited range, including the endpoints. In some embodiments, the use of IgM-depleted enriched LN B cells produces about 10-fold to about 50-fold more IgG-expressing hybridoma clones. In some embodiments, the use of IgM-depleted enriched LN B cells produces about 10-fold to about 25-fold more IgG-expressing hybridoma clones. In some embodiments, the use of IgM-depleted enriched LN B cells produces about 25-fold to about 100-fold more IgG-expressing hybridoma clones. In some embodiments, the use of IgM-depleted enriched LN B cells produces about 50-fold to about 100-fold more IgG-expressing hybridoma clones. In some embodiments, the use of IgM-depleted enriched LN B cells produces about 10-fold to about 75-fold more IgG-expressing hybridoma clones. The amounts can be any value or subrange within the recited ranges, including the endpoints.

[0079] In one aspect, provided herein is an antibody library. In embodiments, the antibody library is prepared using the methods described herein.

[0080] In one aspect, provided herein are antibodies. In embodiments, the antibodies are prepared using the methods described herein.

[0081] In one aspect, provided herein is a hybridoma library. In embodiments, the hybridoma library is prepared using the methods described herein.

[0082] In one aspect, provided herein are kits for preparing the hybridoma or antibody libraries described herein. In embodiments, the kits include at least one adjuvant. In embodiments, the kits include at least two different adjuvants. In embodiments, the kits include reagents for isolating, separating, or enriching B cells (e.g., from other cells in lymph nodes). In embodiments, the kits include beads (microbeads) that interact with at least one reagent. In embodiments, the kits include a column that interacts with at least one reagent. In embodiments, the kits include a fusion partner for generating hybridomas.

[0083] In embodiments, the adjuvant is CFR, Ribi, and / or a TLR agonist cocktail. In embodiments, at least one reagent for isolating, separating, or enriching B cells comprises an antibody specific for B cells (e.g., mouse or rat B cells). In embodiments, at least one reagent for isolating B cells comprises an antibody that recognizes cells other than B cells. In embodiments, the antibody is labeled. In embodiments, the label is attached to a second molecule. In embodiments, the second molecule is attached to a bead or a column. In embodiments, the label is biotin and the second molecule is streptavidin. In embodiments, the beads are magnetic beads. In embodiments, the column is a magnetic column. In embodiments, the fusion partner is engineered to express both surface IgG and secreted IgG.

[0084] Some embodiments relate to devices, apparatus, compositions, formulations, cells, antibodies, adjuvants, hybridomas, populations or pluralities of any of these, and combinations of any of these, that result in the use, production or practice of any of the methods or portions of methods described herein.

[0085] It will be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or variations in light thereof will be suggested to those skilled in the art and are to be included within the spirit and scope of this application and the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. [Example]

[0086] Those skilled in the art will appreciate that the descriptions of making and using the particles described herein are for illustrative purposes only, and that the disclosure is not limited by these examples.

[0087] Example 1. Inbred vs. outbred animals Sprague Dawley rats or Balb / c mice (Charles River) were immunized subcutaneously at the base of the tail with 100 μg of Target A protein (Genentech) mixed with complete Freund's adjuvant (BD Biosciences) or Ribi adjuvant (Sigma-Aldrich), followed by boosts at alternating sites (i.p., bilateral heel joints, or base of the tail) with 50 μg of protein mixed with incomplete Freund's adjuvant (BD Biosciences) or sterile PBS. Serum was collected after five doses and tested by ELISA against the immunizing protein.

[0088] Figure 1A shows that SD rats express higher IgG titers than Balb / c mice. Antigen-specific IgG titers are the dilution ratios that result in half-maximal signals. Target homology is included to demonstrate that the difference in titers is likely not due to differences in antigen tolerance.

[0089] Sprague Dawley rats (Charles River) or C57BL / 6 knockout mice (Genentech) were immunized subcutaneously at the base of the tail with 50 μg of target B protein (Genentech) mixed with complete Freund's adjuvant (BD Biosciences), Ribi adjuvant (Sigma-Aldrich), or a TLR agonist cocktail (as illustrated in Figure 3C). Then, every two weeks, they received boosts with 25 μg of protein mixed with incomplete Freund's adjuvant (Sigma-Aldrich) or sterile PBS at alternating sites (i.p., bilateral heel joints, or base of the tail). Serum was collected after six doses and tested by ELISA against the immunoreactive protein.

[0090] Figure 1B shows that IgG expression titers are higher in SD rats than in C57BL / 6 mice. Antigen-specific IgG titers are the dilution ratios that result in a signal that reaches half the maximum. Target homology is included to demonstrate that the difference in titers is likely not due to differences in antigen tolerance.

[0091] Example 2. Single injection site vs. multiple injection sites Figure 2A shows the location of multiple injection sites for immunization. Base of tail, shoulder, and ankle injections are administered subcutaneously (sc), and antigen-specific B cells are likely to drain into the inguinal and iliac, axillary and brachial, and popliteal lymph nodes, respectively. Intraperitoneal (ip) injections are administered into the peritoneal cavity, and antigen-specific B cells are likely to drain into the mesenteric lymph nodes and spleen.

[0092] Transgenic rats (Open Monoclonal Technology) were immunized weekly with 20 μg of target C or target D protein (Genentech) mixed with Ribi adjuvant (Sigma-Aldrich), either ip alone or at multiple sites alternating every 2 weeks as shown in Figure 2A. Serum was collected after 5 weeks of injection and tested by ELISA against the immunizing proteins.

[0093] Figure 2B shows that antigen-specific titers after single-site immunization were lower than after multiple-site immunization. The antigen-specific IgG titer is the dilution that results in a signal that reaches half the maximum signal.

[0094] Example 3. Twice-Weekly Dosing vs. Once Every 2 Weeks Dosing Figure 3A illustrates the biweekly dosing strategy. This is a general illustration of the expected changes in antigen-specific immune responses upon immunization with an antigen; actual data are not shown. Animals are injected at multiple sites at week 0 with protein combined with an adjuvant (Freund's, Ribi, or TLR agonist cocktail, as described in Example 3) to stimulate a primary immune response as indicated by an increase in serum IgM levels. Animals are then boosted biweekly with smaller doses of antigen, typically diluted in sterile PBS without adjuvant. This stimulates a secondary immune response, resulting in an increase in serum IgG levels and the development of germinal centers for B cell selection and affinity maturation against the target. Serum is collected at multiple time points to assess the progression of the antigen-specific immune response.

[0095] C57BL / 6 knockout mice (Genentech) were immunized twice weekly (every 3–4 days) with 2 μg of target E protein (Genentech) mixed with Ribi adjuvant (Sigma-Aldrich) via the i.p. route, or with 100 μg of target E protein mixed with Ribi adjuvant via the i.p. route, followed by 2-weekly boosts with 50 μg of target E protein mixed with Ribi adjuvant or diluted in sterile PBS via the i.p. route. Serum was collected after 8 or 9 weeks of treatment and tested by ELISA against the immunizing protein.

[0096] Figure 3B shows, surprisingly, that the antigen-specific titers from rats dosed twice weekly were lower than those dosed once every two weeks using Ribi adjuvant. The antigen-specific IgG titer is the dilution that results in a signal that reaches half the maximum signal.

[0097] C57BL / 6 knockout mice (Genentech) were immunized twice weekly (every 3–4 days) with 2 μg of target E protein (Genentech) mixed with a combination of TLR agonists: 50 μg MPL (Sigma-Aldrich), 20 μg R848 (Invivogen), 10 μg Poly I:C (Invivogen), and 10 μg CpG (Invivogen). Alternatively, mice were immunized with 100 μg of target E protein mixed with a TLR agonist cocktail adjuvant, followed by 2-weekly boosts with 50 μg of target E protein mixed with Ribi adjuvant (Sigma-Aldrich) or diluted in sterile PBS. Sera were collected after 8 or 9 weeks of treatment and tested by ELISA against the immunizing proteins.

[0098] Figure 3C shows that the antigen-specific titers from rats dosed twice weekly were lower than those dosed once every two weeks using the TLR agonist-adjuvant mixture. The antigen-specific IgG titer is the dilution that results in a signal that reaches half the maximum signal.

[0099] Example 4. Lymph nodes versus spleen as a source of B cells Figure 4A shows the location of draining lymph nodes near the multiple injection sites. Multiple lymph nodes, including inguinal, iliac, axillary, brachial, mesenteric, and popliteal nodes, are harvested and pooled as a potential source of antigen-specific B cells from animals injected with antigen at multiple sites as described in Figure 2A.

[0100] Transgenic rats (Open Monoclonal Technology) were immunized sc at the base of the tail with 200 μg of target D protein (Genentech) mixed with Freund's complete adjuvant (BD Biosciences) and subsequently boosted with 100 μg of target D protein mixed with incomplete Freund's adjuvant (BD Biosciences) at alternating biweekly sites as shown in Figure 2A, or with 10 μg of target D protein mixed with Ribi adjuvant (Sigma-Aldrich) or a TLR agonist cocktail (described in Example 3) twice a week (every 3–4 days). Three days after the final immunization, approximately 7 weeks after the start of immunizations, spleens or multiple lymph nodes were harvested. B cells from these rats were purified from lymphocytes using magnetic separation (Miltenyi Biotec) as described in Example 6, and 45 million cells from the resulting B cell population were fused with P3X63-Ag8U.1 mouse myeloma cells (American Type Culture Collection) by electrofusion (Harvard Apparatus). The fused cells were incubated overnight at 37°C and 7% CO in Medium C (StemCell Technologies), then resuspended in semi-solid Medium D (StemCell Technologies) containing anti-rat IgG-FITC (Sigma-Aldrich) and plated in Omniwell trays (Thermo Fisher Scientific). Seven days after plating, fluorescent colonies were selected and cultured in Medium E (StemCell Technologies) using a Clonepix FL (Molecular Devices). The supernatants were screened by ELISA against the target D protein 7 days after colony picking.

[0101] Figure 4B shows that the number of antigen-specific hybridoma clones derived from lymph nodes is significantly higher than that derived from spleens. As shown in Figure 4B, obtaining hybridoma clones from spleens produces up to about 10-fold fewer hybridoma clones than obtaining hybridoma clones from lymph nodes. Surprisingly, using the methods described herein, it is possible to obtain 10 times or more the number of antigen-specific IgG+ clones when obtained from lymph nodes rather than spleens.

[0102] Example 5. Parallel use of multiple adjuvants To determine whether mechanical mixing of the CFRs affected the resulting titers, the CFRs were mixed with antigen either mechanically (Omni Mixer, Omni Inc.) or by hand using a syringe. Rats were injected with the adjuvant compositions, and antigen-specific antibody titers were determined as described herein. The results are shown in Figure 5A. As can be seen in Figure 5A, mechanical mixing produces significantly higher antigen-specific serum IgG titers than syringe mixing. The mechanically mixed samples exhibited approximately twice the antigen-specific serum IgG titers of the syringe-mixed samples.

[0103] Transgenic rats (Open Monoclonal Technology) were immunized twice weekly by injection at the base of the tail with 20 μg of target F protein (Genentech) mixed with Ribi adjuvant (Sigma-Aldrich) or the TLR agonist cocktail adjuvant described in Example 3, divided into multiple sites as shown in Figure 2A, or with 150 μg of target F protein mixed with complete Freund's adjuvant (BD Biosciences), followed by biweekly boosts with 50 μg of target F protein mixed with incomplete Freund's adjuvant (BD Biosciences) or 10 μg of CpG (Invivogen), divided into multiple sites as shown in Figure 2A. Serum was collected after 10 weeks of dosing and tested by ELISA against the immunizing protein.

[0104] Figure 5B shows antigen-specific titers from animals immunized with different adjuvants. The antigen-specific IgG titer is the dilution factor that results in a half-maximal signal. As shown in Figure 5B, complete Freund's adjuvant (CFA) has significantly higher antigen-specific IgG titers for this antigen than Ribi adjuvant or TLR agonist cocktail adjuvant, by more than 10 orders of magnitude.

[0105] Transgenic rats (Open Monoclonal Technology) were immunized weekly at multiple sites as shown in Figure 2A with 20 μg of target G protein (Genentech) mixed with Ribi adjuvant (Sigma-Aldrich) or the TLR agonist cocktail adjuvant described in Example 3. Serum was collected after 6 weeks of dosing and tested by ELISA against the immunizing protein.

[0106] Figure 5C shows antigen-specific titers from animals immunized with different adjuvants. The antigen-specific IgG titer is the dilution factor that results in a half-maximal signal. As shown in Figure 5C, the Ribi adjuvant has a significantly higher antigen-specific IgG titer for this antigen than the TLR agonist cocktail adjuvant, approximately 10 times higher.

[0107] Example 6. B cell enrichment and IgM depletion by negative selection Transgenic rats (Open Monoclonal Technology) were immunized sc at the base of the tail with 10 μg of target D protein (Genentech) mixed with Ribi adjuvant (Sigma-Aldrich) and then boosted twice weekly (every 3–4 days) with 10 μg of target D protein mixed with Ribi adjuvant (Sigma-Aldrich) at multiple sites as shown in Figure 2A. Lymph nodes were harvested 3 days after the final immunization, approximately 7 weeks after the start of immunization. Lymphocytes were incubated with anti-rat CD4 (BD Biosciences), anti-rat CD8a (BD Biosciences), anti-rat CD11b / c (BD Biosciences), anti-rat CD161a (BD Biosciences), and anti-rat granulocyte (eBioscience) biotin-conjugated antibodies, washed, and then incubated with streptavidin-coated magnetic beads (Miltenyi Biotec) and run through a magnetic column (Miltenyi Biotec) to capture the unlabeled population. The resulting enriched B cell population was then harvested for fusion or subjected to further depletion steps. Cells were labeled with anti-rat IgM-biotin (BD Biosciences), washed, and then incubated with streptavidin-coated magnetic beads (Miltenyi Biotec) and run through a magnetic column (Miltenyi Biotec) to capture the unlabeled population. Cells from each group were fused with P3X63-Ag8U.1 mouse myeloma cells (American Type Culture Collection) by electrofusion (Harvard Apparatus). Fused cells were incubated overnight at 37°C and 7% CO in Medium C (StemCell Technologies), then resuspended in semi-solid Medium D (StemCell Technologies) containing anti-rat IgG-FITC (Sigma-Aldrich) and plated in Omniwell trays (Thermo Fisher Scientific).Seven days after plating, fluorescent colonies were selected and transferred to 96-well culture plates (BD Biosciences) containing medium E (StemCell Technologies) using Clonepix FL (Molecular Devices). Supernatants were screened by ELISA against target D protein 7 days after colony picking. The number of IgG-positive clones shown is normalized to the fusion of 45 million B cells.

[0108] Figure 6 shows the number of IgG-expressing hybridoma clones derived from whole lymph nodes, enriched LN B cells, and IgM-depleted enriched LN B cells. B cells produced the most numerous IgG-expressing hybridoma clones, followed by enriched LNs. Surprisingly, using the methods described herein, the use of IgM-depleted enriched LN B cells can produce approximately 10- to 100-fold more IgG-expressing hybridoma clones than enriched LN B cells.

[0109] Example 7. Generation of SP2ab vs. PU1 antigen-specific hybridomas Figure 7A: Control hybridoma (left) was analyzed using flow cytometry to identify APC GAM The results are compared with strains transformed with Igα and Igβ after labeling with Ig (right). Histograms show the viable cell population. Hybridomas were generated as described by Harlow and Lane (1988) and grown in culture medium supplemented with HAT (Sigma-Aldrich). Sorted cells were grown in culture medium supplemented with hypoxanthine and thymidine (Sigma-Aldrich). Cells were washed and then stained with allophycocyanin-conjugated goat anti-mouse Ig H+L (Invitrogen). Cells were washed again and resuspended in IMDM (Atlanta Biological Laboratories) containing 20% FBS. Nonviable cells were differentiated from viable cells using propidium iodide or 7-aminoactinomycin D (both Invitrogen). Modified from Price et al., J. Immunol Methods. 2009 March 31;343(1):28-41.

[0110] Figure 7B shows the FACS sorting profile of the SP2ab hybridoma. IgM-negative B cells from immunized rats were purified from lymphocytes using magnetic separation (Miltenyi Biotec) as described in Example 6 and fused with SP2ab mouse myeloma cells (Enzo Life Sciences) via electrofusion (Harvard Apparatus). The fused cells were incubated overnight in medium C (StemCell Technologies) at 37°C and 7% CO2, then plated into 6-well plates containing medium E (StemCell Technologies) supplemented with 1x HAT (Sigma-Aldrich) and incubated for 3 days at 37°C and 7% CO2. Cells were harvested and stained with allophycocyanin-labeled anti-rat IgG (Jackson Immunoresearch) and phycoerythrin-labeled target protein (Genentech). The hybridoma cell population expressed IgG (left histogram), and IgG + Ag + Cells were identified and sorted using a FACSAria Fusion sorter (BD Biosciences) (right dot plot).

[0111] Transgenic rats (Open Monoclonal Technology) were immunized in the base of the tail with 100 μg of target H protein (Biosearch Technologies) mixed with complete Freund's adjuvant (BD Biosciences) and subsequently boosted ip every two weeks with 50 μg of target H protein mixed with incomplete Freund's adjuvant (BD Biosciences). C57BL / 6 knockout mice (Genentech) were immunized twice weekly (every 3–4 days) at multiple sites (intraperitoneally and into both ankle joints) with 2 μg of target I protein (Genentech) mixed with a TLR agonist cocktail (described in Example 3). IgM-negative B cells from immunized animals were purified from lymphocytes using magnetic separation (Miltenyi Biotec) (using the kit for mice or the kit described in Example 6 for rats) and fused with P3X63-Ag8U.1 mouse myeloma cells (American Type Culture Collection) via electrofusion (Harvard Apparatus). The fused cells were incubated overnight at 37°C and 7% CO in Medium C (StemCell Technologies), then resuspended in semi-solid Medium D (StemCell Technologies) containing anti-species IgG-FITC (Jackson Immunoresearch) and plated in Omniwell trays (Thermo Fisher Scientific). Seven days after plating, fluorescent colonies were selected and transferred to 96-well plates containing Medium E (StemCell Technologies) using a Clonepix FL (Molecular Devices). Supernatants were screened by ELISA against the immunizing protein 7 days after picking.

[0112] Figure 7C shows the percentage of antigen-specific hybridomas by IgG-based selection using the PU1 fusion partner. Antigen-positive rates are shown as a function of IgG-expressing wells.

[0113] The same cell populations as above were fused with SP2ab mouse myeloma cells (Enzo Life Sciences) via electrofusion (Harvard Apparatus). The fused cells were incubated overnight in medium C (StemCell Technologies) at 37°C and 7% CO2, then plated into 6-well plates containing medium E (StemCell Technologies) supplemented with 1x HAT (Sigma-Aldrich) and incubated at 37°C and 7% CO2 for 3 days. Cells were harvested and stained with allophycocyanin-labeled anti-rat IgG (Jackson Immunoresearch) and, where appropriate, phycoerythrin-labeled target H or I protein (Genentech). IgG + Ag + Cells were sorted into 96-well plates using a FACSAria Fusion sorter (BD Biosciences). Seven days after plating, supernatants were screened by ELISA against the immunizing protein.

[0114] Figure 7D shows the percentage of antigen-specific hybridomas by antigen-based selection using the Sp2ab fusion partner. Antigen-positive rates are shown as a function of IgG-expressing wells.

[0115] References

[0116] Price, et al. Engineered cell surface expression of membrane immunoglobulin as a means to identify monoclonal antibody-secreting hybridomas. J. Immunol Methods. 2009 March 31;343(1):28-41.

[0117] Harlow, E.; Lane, DP. Antibodies: A Laboratory Manual. CSH Laboratory Press; Cold Spring Harbor, NY:1988.

Claims

[Claim 1] A method for producing an antibody library, comprising: (a) injecting one or more animals with an antigen; (b) harvesting B cell-containing draining lymph nodes from each animal; (c) forming a hybridoma between each B cell and a fusion partner; and (d) screening the hybridomas for binding specificity to the antigen. Including, The following conditions: (i) the animal is an outbred animal; (ii) the animal is injected at multiple sites; (iii) the animals are injected every two weeks; (iv) the animals are injected every 6 to 15 weeks; (v) using multiple adjuvants such that different animals are injected with different adjuvants; (vi) enriching for B cells prior to step (c); and / or (vii) Using a fusion partner engineered to express both surface and secreted IgG The method according to claim 1, wherein at least two of the following are applied:

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