BR-112013001175-B1 - METHOD FOR MAKING A SYNTHETIC POLYPEPTIDE LIBRARY COMPRISING CDRH3 SEQUENCES, SYNTHETIC POLYPEPTIDE LIBRARY AND ITS USE

Abstract

METHOD FOR MAKING SYNTHETIC POLYNUCLEOTIDE LIBRARIES, POLYNUCLEOTIDE LIBRARIES, LIBRARY POLYPEPTIDE EXPRESSION PRODUCTS, ANTIBODY, VECTOR, KIT, HOST CELL AND USE OF SAID LIBRARY. The present invention overcomes the shortcomings inherent in known methods for generating antibody-coding polynucleotide libraries by specifically designing libraries with targeted sequence and length diversity.

Inventors

• Maximiliano Vasquez
• Arvind Sivasubramanian
• Michael Feldhaus

Assignees

• ADIMAB, LLC

Dates

Publication Date

20260310

Application Date

20110714

Priority Date

20100716

Claims (9)

1. 1. A method for making a synthetic polypeptide library comprising CDRH3 sequences, characterized in that it comprises: (a) providing a theoretical segment pool containing TN1, DH, N2, and H3-JH segments; (b) providing a reference set of pre-immune CDRH3 sequences with sequence diversity and length diversity similar to naturally occurring human antibody sequences before those sequences have undergone negative selection and/or hypermutation; (c) using combinations of the TN1, DH, N2, and H3-JH segments contained in the theoretical segment pool of (a) to identify the closest match(es) for each CDRH3 sequence in the reference set of (b); (d) selecting segments from the closest match(es) identified in step (c) for inclusion in a synthetic library; and (e) synthesizing the synthetic CDRH3 library.
2. 2. Method according to claim 1, characterized in that: (a) the segments selected for inclusion in the synthetic library are selected according to their segment usage weight in the CDRH3 sequence reference set, (b) the segments selected for inclusion in the synthetic library are selected according to one or more physicochemical properties, (c) the method further comprises the additional selection of TN1 and N2 segments that occur in the reference set but not in the theoretical segment pool, (d) stop codons are reduced or eliminated from the library, (e) unpaired Cys residues, N-linked glycosylation motifs and deamidation motifs are reduced or eliminated in the translation products of the library, (f) the DH and H3-JH segments are progressively truncated before combination with the CDRH3 sequences in the reference set, (g) the method further comprises introducing one or two degenerate codons into a segment selected from the group consisting of DH and N2, or combinations thereof, or (h) The method also involves introducing a degenerate codon into the H3-JH segment.
3. 3. A library of synthetic polypeptides, characterized in that it comprises 10⁴ polypeptides comprising different CDRH3 sequences with the structure: [TN1]-[DH]-[N2]-[H3-JH], wherein: TN1 is a polypeptide corresponding to any of the TN1 polypeptides in Tables 9-10 and 18-26, or a polypeptide produced by the translation of any of the TN1 polynucleotides in Tables 25-26; DH is a polypeptide corresponding to any of the DH polypeptides in Tables 9, 11, 17-25 and 28, or a polypeptide produced by the translation of any of the DH-encoding polynucleotides in Tables 16, 25 and 27; N2 is a polypeptide corresponding to any of the N2 polypeptides in Tables 9, 12, 18-25 and 30, or a polypeptide produced by the translation of any of the polynucleotides encoding N2 of Tables 25 and 29; and H3-JH is a polypeptide corresponding to any of the H3-JH polypeptides of Tables 9, 13, 15, 18-25 and 32, or a polypeptide produced by the translation of any of the polynucleotides encoding H3-JH of Tables 14, 25 and 31; wherein the library was made using the method as defined in claim 1.
4. 4. Library according to claim 3, characterized in that the CDRH3 polypeptides are produced by the TN1, DH, N2 and H3-JH polypeptide sets provided in any of Tables 23-25.
5. 5. Library according to claim 3, characterized in that CDRH3 polypeptides are produced by the TN1 polypeptide set provided in Table 26, the DH polypeptide set provided in Table 28, the N2 polypeptide set provided in Table 30, and the H3-JH polypeptide set provided in Table 32.
6. 6. Library according to claim 3, characterized in that a number of N-linked glycosylation sites, deamidation motifs and/or Cys residues are reduced or eliminated compared to libraries produced by amplifying a repertoire from a biological source.
7. 7. Library according to claim 3, characterized in that the polypeptides are expressed as full-length IgGs.
8. 8. Use of the library as defined in any one of claims 3 to 7, characterized in that it is for isolating an antibody binding to an antigen.
9. 9. Library according to claim 3, characterized in that the polypeptides are expressed as intact antibodies.

Description

RELATED REQUEST [0001] This application claims priority for U.S. Provisional Application Serial No. 61/365,194, filed July 16, 2010, which is incorporated herein in its entirety by this reference. BACKGROUND [0002] Antibodies have profound relevance as research tools and in diagnostic and therapeutic applications. However, identifying useful antibodies is difficult, and once identified, antibodies generally require considerable reformulation or "humanization" before they are suitable for therapeutic applications in humans. [0003] Many methods for antibody identification involve displaying antibody libraries derived by amplifying nucleic acids from tissues or B cells. Some of these methods have utilized synthetic libraries. However, many of these methods have limitations. For example, most human antibody libraries known in the art contain only the diversity of antibody sequences that can be captured or cloned from a biological source (e.g., B cells) experimentally. In this sense, these libraries may overrepresent some sequences, completely deprive others, or underrepresent others, particularly those binding human antigens. Most synthetic libraries known in the art have other limitations, such as the occurrence of unnatural (i.e., non-human) amino acid sequence motifs that have the potential to be immunogenic. [0004] In this sense, there is a need for diverse antibody libraries containing candidate antibodies that are non-immunogenic (i.e., human) and have desirable properties (e.g., the ability to recognize a wide variety of antigens). However, obtaining these libraries requires balancing the competing goals of generating diverse libraries while still maintaining the human character of the sequences within the library. The present invention provides antibody libraries that have these and other desirable characteristics, and methods for producing and using such libraries. SUMMARY [0005] The present invention provides, among other things, improvements in the design and production of synthetic libraries that mimic the diversity of the natural human repertoire of CDRH3, CDRL3, heavy chain, light chain, and/or full-length (intact) antibody sequences. In some embodiments, the invention defines and provides methods for generating theoretical segment pools of TN1, DH, N2, and H3-JH segments to be considered for inclusion in a physical manifestation of a library (e.g., polynucleotide or polypeptide) comprising or encoding CDRH3 sequences (e.g., an antibody library). In certain embodiments, the present invention defines and provides methods for matching the individual elements of these theoretical segment pools to a reference set of CDRH3 sequences, to determine the frequency of occurrence (or segment usage weight) of each of the segments in the theoretical segment pool in the reference set. While any set of CDRH3 sequences can be used as a reference set, the invention also defines and provides methods for generating specific reference sets or subsets of interest. For example, among other things, the present invention provides methods for filtering an original defined reference to obtain a reference set provided with a pre-immune character. Furthermore, methods are provided for defining and/or identifying segments that occur within CDRH3 sequences in the reference set but not in the theoretical segment pool. Such segments can be added to a theoretical segment pool, for example, to be considered for inclusion in a physical library. Although the frequency of occurrence of a given segment of a reference set is useful for selecting segments for inclusion in a physical library, the invention also provides a number of physicochemical and biological properties that can be used in conjunction (alone or with any other criteria) to select segments for inclusion in a physical library. [0006] In some embodiments, the invention provides libraries that differ from some other libraries known in the art in that they are not stochastic from place to place in composition or sequence and are therefore inherently less random than those other libraries known in the art (see, for example, Example 14 of US Pub. No. 2009/0181855, incorporated by reference in its entirety, for a discussion of information content and randomness). In some embodiments, degenerate oligonucleotides can be used to increase the diversity of elements in a library while additionally improving the match to a reference set of sequences (e.g., CDRH3, CDRL3, heavy chain, light chain, and/or full-length (intact) antibody sequences). [0007] The invention also provides libraries whose elements have sequences that relate to each other, wherein these would be selected for inclusion in a physical library by performing the analyses described in this document, for example, generating a defined CDRH3 reference as in Example 3; generating theoretical segment pools as in Examples 5 to 7; matching the elements of a theoretical segment pool to the defined reference match as in Example