Rabbit hybridoma

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A rabbit hybridoma is a hybrid cell line formed by the fusion of an antibody producing rabbit B cell with a cancerous B-cell (myeloma).

History

The rabbit immune system has been documented as a vehicle for developing antibodies with higher affinity and more diverse recognition of many molecules including phospho-peptides, carbohydrates and immunogens that are not otherwise immunogenic in mouse.[1] However, until recently, the type of antibodies available from rabbit had been limited in scope to polyclonal antibodies. Several efforts were made to generate rabbit monoclonal antibodies after the development of mouse hybridoma technology in the 1970s.[2] Research was conducted into mouse-rabbit hetero-hybridomas to make rabbit monoclonal antibodies.[1][3] However, these hetero-hybridomas were ultimately difficult to clone, and the clones, generally unstable, and did not secrete antibody over a prolonged period of time.

Initial fusion partner

In 1995, Katherine Knight and her colleagues, at Loyola University of Chicago, succeeded in developing a double transgenic rabbit over-expressing the oncogenes v-abl and c-myc under the control of the immunoglobulin heavy and light chain enhancers. The rabbit formed a myeloma-like tumor, allowing the isolation of a plasmacytoma cell line, named 240E-1. Fusion of 240E-1 cells with rabbit lymphocytes produced hybridomas that secreted rabbit monoclonal antibodies in a consistent manner.[4] However, like the early mouse myeloma lines developed in the 1970s, stability was a concern. A number of laboratories which had received the 240E-1 cell line from Dr. Knight’s laboratory reported stability problems with the fusion cell line 240E-1.[5]

Improved fusion partner

In 1996, Weimin Zhu and Robert Pytela, at the University of California San Francisco (UCSF), obtained 240E-1 from Dr. Knight’s laboratory and attempted to develop an improved rabbit hybridoma.[4] Improvements in the characteristics of 240E-1 were accomplished by repeated subcloning, selection for high fusion efficiency, robust growth, and morphological characteristics such as a bright appearance under a phase-contrast microscope. Selected subclones were further tested for their ability to produce a stable hybridoma and monoclonal antibody secretion. After multiple rounds of subcloning and selection processes, a new cell line named 240E-W, was identified and which expressed better fusion efficiency and stability. Cell line 240E-W has since been further developed and optimized for production of rabbit monoclonal antibodies for research and commercial applications.

Process

The process of hybridoma formation in a rabbit first entails obtaining B-cells from a rabbit that has been immunized. There are numerous immunization protocols for rabbit, notably for the generation of polyclonal antibodies.[6][7][8] After immunization, B-cells are fused with a candidate rabbit fusion partner cell line to form hybridomas. Resulting antibodies from hybridomas are screened for an antigen which meets criteria of interest by diagnostic tests such as ELISA, western blot, immunohistochemistry, and FACS. The resulting hybrdomas may be subcloned to ensure monoclonal characteristics.

Humanization of rabbit antibodies

Mitchell Ho and Ira Pastan at National Cancer Institute (Bethesda, USA) isolated a group of rabbit monoclonal antibodies (e.g. YP218, YP223) that recognize rare epitopes of mesothelin, including poorly immunogenic sites close to the C terminal end, for cancer therapy. [9] Dr. Ho's laboratory analyzed the complex structures of rabbit antibodies with their antigens from the Protein Data Bank, and identified antigen-contacting residues on the rabbit Fv within the 6 Angstrom distance to its antigen.[10] They named "HV4" and "LV4", non-complementarity-determining region (CDR) loops that are structurally close to the antigen and located in framework 3 of the rabbit heavy chain and light chain, respectively. Based on computational structural modeling, Ho and Zhang designed a humanization strategy by grafting the combined Kabat/IMGT/Paratome CDRs into a human germline framework sequence. The immunotoxins composed of the humanized rabbit Fvs (e.g. hYP218) fused to a clinically used toxin showed stronger cytotoxicity against tumor cells than the immunotoxins derived from their original rabbit Fvs. The CAR T cells based on the hYP218 antibody also show effective inhibition of tumor growth in mice. [11] The method (i.e. grafting combined Kabat/IMGT/Paratome rabbit CDRs to a stable human germline framework) has been suggested as a general approach to humanizing rabbit antibodies.[10]

References

  1. ^ a b Raybould TJ, Takahashi M (June 1988). "Production of stable rabbit-mouse hybridomas that secrete rabbit mAb of defined specificity". Science. 240 (4860): 1788–90. Bibcode:1988Sci...240.1788R. doi:10.1126/science.3289119. PMID 3289119.
  2. ^ Collins JJ, Black PH, Strosberg AD, Haber E, Bloch KJ (February 1974). "Transformation by simian virus 40 of spleen cells from a hyperimmune rabbit: evidence for synthesis of immunoglobulin by the transformed cells". Proceedings of the National Academy of Sciences of the United States of America. 71 (2): 260–2. Bibcode:1974PNAS...71..260C. doi:10.1073/pnas.71.2.260. JSTOR 62751. PMC 387981. PMID 4150020.
  3. ^ Kuo MC, Sogn JA, Max EE, Kindt TJ (April 1985). "Rabbit-mouse hybridomas secreting intact rabbit immunoglobulin". Molecular Immunology. 22 (4): 351–9. doi:10.1016/0161-5890(85)90119-1. PMID 4033662.
  4. ^ a b Spieker-Polet H, Sethupathi P, Yam PC, Knight KL (September 1995). "Rabbit monoclonal antibodies: generating a fusion partner to produce rabbit-rabbit hybridomas". Proceedings of the National Academy of Sciences of the United States of America. 92 (20): 9348–52. Bibcode:1995PNAS...92.9348S. doi:10.1073/pnas.92.20.9348. PMC 40982. PMID 7568130.
  5. ^ Liguori MJ, Hoff-Velk JA, Ostrow DH (June 2001). "Recombinant human interleukin-6 enhances the immunoglobulin secretion of a rabbit-rabbit hybridoma". Hybridoma. 20 (3): 189–98. doi:10.1089/027245701750293529. PMID 11461668.
  6. ^ Howard GC, Kaser MR, eds. (2007). Making and using antibodies: a practical handbook. CRC Press. ISBN 9780849335280.
  7. ^ Harlow E, Lane D (1988). Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory. ISBN 978-1-936113-81-1.
  8. ^ Coligan JE, Kruisbeek AM, et al., eds. (1994). Current protocols in immunology. Greene and Wiley.
  9. ^ Zhang YF, Phung Y, Gao W, Kawa S, Hassan R, Pastan I, Ho M (May 2015). "New high affinity monoclonal antibodies recognize non-overlapping epitopes on mesothelin for monitoring and treating mesothelioma". Scientific Reports. 5: 9928. Bibcode:2015NatSR...5E9928Z. doi:10.1038/srep09928. PMC 4440525. PMID 25996440. Material was copied from this source, which is available under a Creative Commons Attribution 4.0 International License.
  10. ^ a b Zhang YF, Ho M (April 2017). "Humanization of rabbit monoclonal antibodies via grafting combined Kabat/IMGT/Paratome complementarity-determining regions: Rationale and examples". mAbs. 9 (3): 419–429. doi:10.1080/19420862.2017.1289302. PMC 5384799. PMID 28165915. Public Domain This article incorporates text from this source, which is in the public domain.
  11. ^ Zhang Z, Jiang D, Yang H, He Z, Liu X, Qin W, et al. (June 2019). "Modified CAR T cells targeting membrane-proximal epitope of mesothelin enhances the antitumor function against large solid tumor". Cell Death & Disease. 10 (7): 476. doi:10.1038/s41419-019-1711-1. PMC 6572851. PMID 31209210.

External links

External links

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